DD-Methionine and gold chloride alleviate adverse effects of glutamate on

motility of ephyrae of Aurelia aurita (Linnaeus, 1758) (Scyphozoa:

Semaeostomeae)

D.B. Spangenberg1,*, F.A. Lattanzio2,* & G. Navarro11Department of Pathology and Anatomy, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501, USA2Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA 23501, USA

(*Authors for correspondence: Tel.: +1-757-446-5626, Fax: +1-757-446-5719, E-mail: spangedb@evms.edu(Spangenberg); Tel.: +1-757-446-5636, Fax: +1-757-624-2270, E-mail: lattanfa@evms.edu (Lattanzio))

Key words: jellyfish, pulsing, swimming

Abstract

Glutamate (MSG) causes low pulse numbers and swimming cessation in Aurelia jellyfish ephyrae. Ephyraegiven MSG for 1 h and subsequently maintained in artificial sea water (ASW) were observed at 1, 3, 24, and48 h intervals. Abnormality of motility was found at all post-treatment periods but some ephyrae resumedswimming and normal pulsing within 48 h. Swimming and pulsing were impaired in a significant number ofephyrae within 15 min of MSG treatment. The mechanism of MSG action on ephyrae motility is unknown,but glutamate damage to neurons and hair cells of higher animals is partly attributed to the formation ofreactive oxygen species (ROS). Laser confocal fluorescent microscopy of ephyrae following MSG treatmentindicated an increase of calcium and free radicals in the ephyrae as early as 5 min following MSG exposure.To determine whether antioxidants could alleviate MSG effects, we exposed ephyrae to gold chloridebefore, during, and after treatment with MSG. Ephyrae given gold chloride pre-treatment for 1 h and thentransferred into gold chloride plus MSG for 1 h showed statistically significant recovery from MSGimpairment of pulsing at the 3, 24, and 48 h post-glutamate time periods and higher numbers of swimmersat 3 h and 24 h. Ephyrae groups given gold plus MSG but without gold pretreatment showed recovery ofswimming at 24 h and pulsing at 48 h. DD-methionine given simultaneously with MSG significantly im-proved the pulse numbers and swimming of ephyrae at the 3, 24, and 48 h post-glutamate time periodscompared to those receiving MSG alone. Both DD-methionine and gold chloride accelerated the time ofrecovery from glutamate-induced motility impairment, possibly through their antioxidant activities.

Introduction

In mammals, including humans, a cascade ofevents generated by glutamate excitotoxicity is in-volved in brain and spinal cord trauma, aging, andneurodegenerative diseases, including stroke, ALS,Parkinson’s, and Alzheimer’s diseases. One of theearliest events in glutamate excitotoxicity is themodification of glutamate receptors, including N-methyl-DD-aspartate (NMDA), which results in aninflux of calcium into neurons followed by othercellular events leading to neuronal necrosis and/or

apoptosis. The generation of free radicals includingreactive oxygen species (ROS) following excessiveglutamate exposure is believed to play an impor-tant role in the subsequent neuronal damage.

Very little is known concerning the effects ofexcessive glutamate on lower organisms, especiallythe coelenterates. We therefore use tiny ephyrae ofAurelia aurita (Linnaeus, 1758) to study effects ofextraneously introduced monosodium gluta-mate (MSG) on their pulsing and swimmingmotility. Ephyrae respond rapidly, adversely, andconsistently to glutamate treatment, allowing study

Hydrobiologia 530/531: 355–363, 2004.D.G. Fautin, J.A. Westfall, P. Cartwright, M. Daly & C.R. Wyttenbach (eds),Coelenterate Biology 2003: Trends in Research on Cnidaria and Ctenophora.� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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of compounds that may prevent such responses. Todetermine whether substances which reportedlyhave free radical scavenging abilities will negate theadverse effects of glutamate, we introduced goldchloride and (separately) DD-methionine with glu-tamate to the ephyrae. Further, using fluorometricdyes and laser confocal microscopy, we endeavoredto demonstrate whether there is an increase inintracellular calcium activity and the generation ofincreased free radicals in the ephyrae shortly aftertheir exposure to glutamate.

Material and methods

Testing

At least three experiments were done at differenttimes for each of the compounds tested, using two-four ephyrae per test group per experiment. Polypswere exposed to 10)5 M iodine in artificial seawater (ASW) to produce ephyrae through strobi-lation of the polyps (Spangenberg, 1967). Testephyrae were selected at least 2 days after releasefrom their strobilae when they are able to swimwell. All test compounds were dissolved in theASW used to culture the jellyfish (Spangenberg,1965). Ephyrae were placed in test solutions in newglass test tubes, previously washed in distilledwater, and dried. Each tube held one ephyra. Theephyrae were maintained at room temperature(16–19 �C) and were not fed during the testing.

Compounds tested

Tests were done to determine the tolerance ofephyrae to MSG using a series of MSG concen-trations in ASW. From these tests, the concen-tration of 5 mM MSG was selected because itelicited the most consistent response from the jel-lyfish, was non-lethal, and permitted recovery withtime. Similarly, a series of tests was done todetermine the appropriate dosage of gold chloride(0.022 lM) and DD-methionine (5 mM) for use inconjunction with MSG. All chemicals were pur-chased from Sigma (St. Louis, MO).

At intervals of baseline (prior to addition oftest compounds), 1 h in the test compound, and 1,3, 24, and 48 h after removal from the test com-pound, the test was read as follows: Each test tube

was inverted to bring the ephyra into the upperportion of the tube and the ephyra was observedfor one minute. The number of pulses per minutewas counted using a manual counter and the ani-mal’s swimming behavior was noted and recorded.Ability to swim was defined as the ability of theephyra to move with direction along linear orcurvilinear, but not circular pathways, againstcurrent or gravity. A normal pulse was defined as arapid simultaneous contraction of all lappetsdownward at an angle of >80�, resulting inmovement of the ephyra.

Statistics

Test results were subjected to the Student’s pairedt-test and the nonparametric Rank Sum test todetermine statistical significance, with p < 0.05.Sample number (n) ranged from 6 to 60 ephyrae.

Laser confocal fluorescent microscopy studies

Laser confocal fluorescent microscopy was used todetect calcium and free radicals in the ephyraeduring the first hour after exposure of the ephyraeto MSG. We evaluated several visible light calciumindicators, including fura red, fluo-3, and rhod-2.Fluo-3 and rhod-2 were suitable, but fura red hadpoor cellular loading characteristics and was notused. The free radical indicator 5-(and 6-)carboxy-2¢,7¢ dichlorodihydrofluorescein diacetate (car-boxy-H2DCFDA), which is sensitive to peroxide,hydroxyl, and nitric oxide (NO) amongother ROS, was also used, as was DAF-FMdiacetate (4-amino-5-methylamino-2¢,7¢-difluores-cein), a more specific NO indicator. All fluorescentreagents were obtained from Molecular Probes(Eugene, OR). Combinations of 5 lM fluo-3,5 lM rhod-2 AM (acetoxymethyl ester), 10–20 lM carboxy-H2DCFDA, and 10 lM DAF-FM diacetate with 0.01% Pluronic 127 in ASWwere incubated with ephyrae for 1–2 h at 22 �C inglass vials in the dark, with approximately 1 mlsolution per ephyra. After transfer into ASW andwashout of the residual indicator for 15–30 min,ephyrae were placed on a petri dish with a centralwell with a 12 mm glass cover slip bottom (WorldPrecision Instruments, Sarasota, FL) and wererestrained with a fine nylon mesh held over theephyrae using a stainless steel washer. This

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arrangement restricted the movement of the ani-mals and also permitted exchange of bathingsolutions. The ephyrae were not impaired by thetreatments as determined by both microscopicmeasurements and motility tests. Temperatureduring these experiments was 22–25 �C.

Fluorescence measurements were made using aZeiss 510 laser confocal microscope. Appropriateexcitation wavelength and emission filter and di-chroic combinations permitted simultaneousmeasurements of intracellular calcium, ROS, andNO using fluo-3, carboxy-H2DCFDA, and DAF-FM diacetate, respectively (green images collectedat 488 nm excitation, 505–550 nm emission) andintracellular calcium activity (red images collectedat 543 nm excitation 560–615 nm emission) usingrhod-2, as well as single wavelength experimentswith individual indicators. Pinhole settings werenominally 1 Airey unit for the selected objective.Microscope objectives used are listed in the figurelegends. An ASW solution containing 10 lMionomycin was used to saturate the calcium indi-cators at the end of the experiment.

Using a Zeiss 10·, 25·, or 40· Plan NeoFluorlens, 2–5 lm optical slices of the lappet andrhopalia were taken. Depending on the thicknessof the ephyrae or rhopalia and the optical section,15–30 512 · 512 pixel, 1024 bit resolution imageswere collected per time point and stored as multi-image TIFF files. Fluorescence for each indicatorwas measured using the Metamorph image pro-cessing program (Universal Imaging, Downing-town, PA) by first color separating the images andthen determining the intensity of six areas whoseposition was maintained constant during theexperiment. This system permitted confocal mea-surements of ephyrae using low power (to 40·objectives) but was not suitable for higher mag-nifications due to the large working distancecaused by the well slide’s and ephyrae’s thickness.

Results

Glutamate dosage range

Testing a range of MSG from 0.5 to 5 mM re-vealed that ephyrae tolerated a dosage of 5 mMfor up to 24 h. As compared with baseline andsame time period controls, ephyrae given 5 mM

for 24 h were more severely impaired with regardto both pulsing and swimming ability than thosereceiving lower dosages (data not shown). Furthertesting revealed that exposure to 5 mM MSG for1 h is sufficient to impair the pulsing and swim-ming motility of ephyrae as compared with same-time ASW controls. We therefore designed a seriesof tests in which the ephyrae were treated for 1 hin 5 mM MSG in the presence of either goldchloride (0.022 lM) or DD-methionine (5 mM),followed by transfer of the ephyrae to either ASWor ASW with gold, as noted in Tables 1 and 2.

Gold chloride

Ephyrae pre-treated for 1 h with gold chloridebefore exposure to MSG plus gold chloride for 1 hshowed improvement in pulse numbers/min ascompared with ephyrae from MSG treatmentalone at 3, 24, and 48 h after transfer from theMSG into ASW or ASW + gold chloride. With-out gold preteatment, administration of goldchloride simultaneously with MSG generally pro-vided no significant protection from pulsingreduction, although a trend was present and wasmanifested at 48 h in the case of the gold + MSGinto ASW group (see Table 1). Only the goldpretreatment groups eventually recovered pulsingto the point of not being statistically different fromthe ASW control group.

The number of ephyrae that could swim afterreceiving gold chloride pretreatment for 1 h priorto MSG treatment as well as groups receiving goldsimultaneously with MSG for 1 h was significantlyhigher than the MSG alone group at periods of 3and 24 h or 24 h post-MSG treatment, indicatingthat the gold chloride alleviated the swimmingimpairment caused by MSG in some ephyrae. Thenumber of ephyrae swimming in all the MSG-treated gold groups was significantly lower thanASW controls, except those at the 24 h time periodthat had been given gold chloride prior to andsimultaneously with MSG and transferred toASW + gold chloride.

DD-Methionine

Ephyrae given DD-methionine simultaneously withMSG for 1 h had significantly higher pulse num-bers/min than ephyrae given MSG alone at 3, 24,

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and 48 h post-MSG, but were significantly lessactive at the 1 h in and 1 h post-MSG time peri-ods. A significantly higher number of theDD-methionine plus MSG treated ephyrae swam ascompared with those receiving glutamate alone inthe 3, 24, and 48 h post-MSG time periods.Ephyrae given DD-methionine alone for 1 h main-

tained pulsing levels equivalent or better thanASW controls throughout the experiment. As wasshown also with the gold chloride studies, MSGwhen given alone caused a statistically significantreduction in the number of ephyrae able to swimand to pulse as compared with ASW controls at allbut the 45 min pulsing measurement.

Table 1. Effects of antioxidants and MSG on ephyrae pulsing rates (pulses per minute)

Base 15 min 30 min 45 min 60 min 1 h out 3 h out 24 h out 48 h out

MSG

Mean 107.0 61.7* 68.5* 92.3 46.0* 23.7* 16.2* 13.2* 9.6*

Std dev 46.0 33.6 39.9 36.0 41.7 26.7 13.5 35.0 20.3

n 46 22 11 11 35 32 24 24 18

ASW

Mean 96.2 90.4 108.5 104.9 97.4 94.4 79.3 79.9 86.5

Std dev 40.4 36.1 47.3 44.9 34.8 36.2 31.6 29.8 42.5

n 60 24 13 13 49 44 36 36 24

Met

Mean 95.3 115.8 89.4 104.6* 79.4 74.8

Std dev 33.8 21.3 29.2 27.3 32.0 33.4

n 12 n.d. n.d. n.d. 12 12 12 12 12

MSG + Met

Mean 97.2 17.5* 6.8* 41.0* 63.0 82.3

Std dev 23.0 8.6 4.9 36.1 52.6 39.7

n 12 n.d. n.d. n.d. 12 12 12 12 12

PreAu/MSG + Au�Au

Mean 92.2 49.9* 19.1* 29.6* 64 51.2

Std dev 38.6 45.7 14.8 30.6 54.9 58.4

n 12 n.d. n.d. n.d. 12 12 12 12 6

PreAu/MSG + Au�ASW

Mean 102.5 53.9* 23.8* 30.8* 46.7* 65.0

Std dev 42.9 56.8 28.0 28.5 55.4 61.5

n 12 n.d. n.d. n.d. 12 12 12 12 6

MSG + Au�Au

Mean 115.2 39.3* 15.3* 28.3* 30.8* 30.2*

Std dev 50.1 44.4 12.4 31.3 29.4 38.7

n 12 n.d. n.d. n.d. 12 11 12 12 6

MSG + Au�ASW

Mean 98.5 41.0* 42.7* 19.0* 20.3* 41.0*

Std dev 45.5 36.9 55.4 18.0 17.2 36.9

n 12 n.d. n.d. n.d. 12 12 12 12 12

*Different from corresponding ASW value, p < 0.05.

Bold different from corresponding MSG value, p < 0.05.

n.d. – no data.

MSG = 5 mM MSG exposure for 1 h, agents added as noted; ASW = artifical seawater; Met = 5 mM DD-methionine (1 h);

MSG + Met = 5 mM MSG and 5 mM DD-methionine (1 h); PreAu/MSG + Au/Au = 1 h pretreatment with 0.022 lM gold

chloride, followed by gold + MSG (1 h), then placed in gold chloride; PreAu/MSG + Au/Au = pretreatment with gold chloride,

followed by gold + MSG (1 h), then placed in ASW; Au + MSG/Au = gold chloride + MSG (1 h) then placed in gold chloride;

Au + MSG/ASW = gold chloride and MSG (1h) then placed in ASW.

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Glutamate short-term exposure

Tests were done to determine the early effects ofMSG on ephyra swimming and pulsing. Ephyraewere given 5 mMMSG and observed every 15 minup to 1 h and 1 h post-treatment for pulsing andswimming effects as compared with ASW controls.We found that swimming is impaired in a signifi-cant number of ephyrae within 15 min ofMSG treatment and the impairment persiststo 48 h post-MSG administration. Pulsingnumbers were significantly lower as comparedwith ASW controls at 15, 30, 60 min, and 1 hpost-MSG. The maintenance of pulsing at 45 minmay reflect a neurotoxic stimulation of the ephy-rae’s pacemaker prior to the onset of glutamatetoxicity.

Fluorometric studies

Fluorometric studies of calcium and ROS inephyrae revealed an initial increase in calcium andespecially ROS in the ephyrae, with a specific ROSincrease in the rhopalia (Fig. 1A and B). In Fig.1C and D, the presence of baseline NO in therhopalia is demonstrated. At higher magnification,over 30–40 min post-MSG, the area and intensityof the calcium signal within cells of the rhopaliaappear to be increasing (Fig. 1E and F) compared

to control cells (Fig. 1C and E). Specific increasesin NO, as well as ROS, also occurred in somerhopalia (Fig. 1D) as determined by DAF-FMfluorescence. A plot of the rhopalia rhod-2 andcarboxy-H2DCFDA fluorescence confirms statis-tically significant increases in intracellular calciumand ROS activity over time following 5 mM MSGtreatment (Fig. 2).

Discussion

Using Aurelia ephyrae, we have shown thatextraneously administered glutamate causes a de-creased pulsing rate and inhibition of swimming.Swimming cessation and pulsing reduction occuras early as 15 min after administration of theglutamate in many ephyrae. It is not known whe-ther glutamate functions as a neurotransmitter inAurelia, but the effect of glutamate on ephyraswimming strongly suggests that glutamate couldhave neurotransmitter functions, especially inneurons of the neuromuscular system and pace-makers. Further, glutamate could be affecting ep-hyra hair cell synapses.

The glutamate results also suggest that ephyraehave specific glutamate receptors which may befound also in other coelenterates. In Hydra, Kass-Simon & Scappaticci (2004) found that glutamatecauses a significant increase in the number of

Table 2. Effects of antioxidants and MSG on ephyrae swimming

Base 15 min 30 min 45 min 60 min 1 h out 3 h out 24 h out 48 h out

MSG swimmers/total 46/47 15/23* 2/11* 2/11* 2/35* 1/32* 1/24* 1/24* 2/18*

ASW swimmers/total 59/59 23/23 13/13 12/13 49/49 44/44 46/46 36/36 24/24

Met swimmers/total 13/13 n.d. n.d. n.d. 13/13 13/13 13/13 13/13 13/13

MSG + Met swimmers/total 12/12 n.d. n.d. n.d. 4/12* 0/12* 7/12* 8/12 12/12

PreAu/MSG + Au�Au swimmers/total 12/12 n.d. n.d. n.d. 6/12* 3/12* 7/12* 8/12 2/6*

PreAu/MSG + Au�ASW swimmers/total 12/12 n.d. n.d. n.d. 4/12* 3/12* 6/12* 6/12* 3/6

MSG + Au�Au swimmers/total 12/12 n.d. n.d. n.d. 1/12* 0/12* 3/12* 7/12* 3/6

MSG + Au�ASW swimmers/total 12/12 n.d. n.d. n.d. 1/12* 0/12* 1/12* 5/12* 1/6*

*Different from corresponding ASW value, p < 0.05.

Bold different from corresponding MSG value, p < 0.05.

n.d. – no data.

MSG = 5 mM MSG exposure for 1 h, agents added as noted; ASW = artifical seawater; Met = 5 mM DD-methionine (1 h);

MSG + Met = 5 mM MSG and 5 mM DD-methionine (1 h); PreAu/MSG + Au/Au = 1 h pretreatment with 0.022 lM gold

chloride, followed by gold + MSG (1 h), then placed in gold chloride; PreAu/MSG + Au/Au = pretreatment with gold chloride,

followed by gold + MSG (1 h), then placed in ASW; Au + MSG/AU = gold chloride + MSG (1 h) then placed in gold chloride;

Au + MSG/ASW = gold chloride and MSG (1 h) then placed in ASW.

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stenoteles (nematocysts) responding to directmechanical stimuli. The effect was mimicked byNMDA together with kainate or NMDA with

AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid). These authors concluded that aglutamatergic mechanism working through iono-

Figure 1. Note that due to movement of the ephyrae, there were changes in the orientation of the lappets and rhopalia between

measurements. (A) Control rhopalium with rhod-2 AM and carboxy-H2DCFDA, seen with 10· objective. (B) Same rhopalium as A

after 15 min exposure to 5 mm MSG. Note increase in ROS fluorescence in rhopalium and in surrounding lappet. Measurements also

confirmed increase in rhod-2 signals in rhopalium. (C and D) Control rhopalia of two ephyrae loaded with rhod-2 and DAF-FM

diacetate, seen with 25· objective. (E) 30 min after 5 mM MSG and seen with the 25· objective, the rhopalium in C shows increased

calcium fluorescence. (F) 32 min after exposure to 5 mm MSG and seen with the 40· objective, the rhopalium of D shows increases in

calcium and NO.

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tropic glutamate receptors appears to lower thefiring threshold of stenoteles (which are inner-vated), perhaps by permitting the entry of Ca2+

into the cells through the early evolved NMDA/kainate/AMPA mechanism. Bellis et al. (1991) hadidentified two distinct binding sites for LL-glutamicacid in a crude membrane fraction from Hydravulgaris. Also, Smith et al. (2004) found andcloned a glutamine synthetase gene from the seaanemone Aiptasia pallida.

In mammals, glutamate has neurotransmitterfunctions. Glutamate excitotoxicity, which occursas a result of excessive glutamate, has been de-scribed as causing increasing damage of cell com-ponents, including mitochondria, leading to celldeath. In the process, ROS are generated (Atlanteet al., 2001). During the course of the cell damage,increases in O�

2 and hydrogen peroxide (H2O2) oc-cur (Ishikawa et al., 1999). These authors reportedthat both H2O2 and O�

2 play important roles in theresultant apoptosis in embryonic hippocampalneurons in culture. Additionally, NO and/or per-oxynitrite are involved in the cell destruction.

Fluorometric studies of calcium, ROS,and NOS in ephyrae revealed increased calcium-,ROS-, and NOS-generated nitric oxide (NO) in theephyrae as early as 5 min, with increases up to50 min after glutamate treatment (data not

shown). This result suggests that swimmingimpairment is associated with the calcium influxand ROS increases, which are early events in glu-tamate excitotoxicity in mammals.

A number of antioxidants reduce glutamateexcitotoxicity effects (Miyamoto et al., 1989;Mazzio et al., 2001), including DD-methionine andgold compounds. In Aurelia, ephyrae givenDD-methionine simultaneously with MSG for 1 hhad significantly higher pulse numbers/min thanephyrae given MSG alone at 3, 24, and 48 h post-MSG. More ephyrae, treated simultaneously withDD-methionine and MSG for 1 h, were able to swimat all times from 3 h post-glutamate treatment ascompared with those receiving MSG alone.DD-methionine protects against cisplatin-inducedhearing loss when co-administered or adminis-tered prior to cisplatin in the Wistar rat (Camp-bell et al., 1996; Reser et al., 1999). According toSmoorenburg et al. (1999), DD-methionine, whichis a thiol compound, may protect againstcis-platin-induced hearing loss through the scav-enging of cisplatin-induced free radicals.DD-methionine also protected against gentamicin-induced free radical generation in a non-enzy-matic system in vitro and in the guinea pig in vivo,according to Sha & Schacht (2000).

Gold chloride also may function as an anti-oxidant in Aurelia ephyrae. Gold-chloride-treatedephyrae pre-treated for 1 h before exposure toMSG plus gold chloride showed improvement inpulsing as compared with ephyrae from MSGtreatment alone at 3, 24, and 48 h after transferfrom the MSG into ASW or ASW + gold chlo-ride. The number of ephyrae that could swim afterreceiving gold chloride for 1 h prior to MSGtreatment as well as simultaneously with MSG wassignificantly higher than the number of ephyraeswimming after treatment with MSG alone at 3and 24 h post-MSG treatment, indicating that thegold chloride alleviated the swimming impairmentcaused by MSG in some ephyrae. Improvementin pulsing and swimming ability may be directlyassociated with recovery of motor and/or pace-maker neurons damaged by MSG. Richardset. al. (2002) recognized the need for moreresearch of the neuropharmacology and neu-rochemistry of gold.

Many therapeutic gold-containing drugs aregold (I) compounds whereas gold chloride is a gold

0

20

40

60

80

100

120

140

160

Control 5min MSG 10minMSG

15minMSG

Rel

ativ

e flu

ores

cenc

e

ROSCalcium

Figure 2. Elevation of intracellular calcium and ROS in ephy-

rae treated with 5 mM MSG. Ephyrae were loaded with rhod-2

AM and carboxy-H2DCFDA, treated with 5 mm MSG, and

their fluorescence determined as described in the Methods sec-

tion. Results of seven measurements were combined and ana-

lyzed by paired t-test, with the 5, 10, and 15 min time points

found statistically significantly increased ( p < 0.05) from time

0 values.

361

(III) compound. Gold (I) drugs selectively activatethe DNA binding of a heterodimer consisting ofthe basic-leucine zipper transcription factors Nrf2and Maf (Kataoka et al., 2001). Once bound to itsrecognition DNA sequence termed antioxidant-responsive element, Nrf1/Maf induces a set ofantioxidant stress genes whose products contributeto the scavenging of ROS and exhibit anti-inflammatory effects. Graham et al. (1993) notedthat aurocyanide, which is a potent inhibitor of theoxidative burst of neutrophils, may be metabolizedto Au(III) complexes.

Other gold drug studies emphasized the use ofactivated white blood cells, which produce super-oxide ions when activated, to determine effective-ness of gold drugs in reducing the levels of theseO�

2 ions. Auranofin (Hafstrom et al., 1984) andaurothiomalate and triethylphosphine (Daviset al., 1983) are among gold compounds effectivein inhibiting superoxide radical production. Morerecently, Aaseth et al. (1998) noted that thera-peutically given gold salts, through a lysosomalloading of the metal, reduce toxic oxygen pro-duction.

Klein & Ackerman (2003) stated that themechanisms by which neurons die under condi-tions of oxidative stress remain largely unknown.These authors point out that excessive ROS canlead to the destruction of cellular componentsincluding lipids, protein, and DNA, and ultimatelycell death is via apoptosis or necrosis. Glutamateneurotoxicity has been linked to mitochondrialuptake of Ca2+ as well as to the activation ofcalmodulin and neuronal nitric oxide synthase inmotor neurons (Urushitani et al., 2001). Fluoro-metric analysis showed that mitochondrial Ca2+

was elevated promptly following exposure to0.5 mM glutamate for 5 min with subsequentaccumulation of ROS in the mitochondria. TheNMDA receptor was also activated. Fluorometricanalyses of free radicals in ephyrae followingtreatment with 5 mM MSG indicate an increase offree radicals, including NO, in the ephyrae as earlyas 5 min following treatment. Vergun et al. (2001),using inhibitors of lipid peroxidation and scav-engers of superoxide or hydrogen peroxide duringand following a 10 min exposure to glutamate,found that the antioxidants delayed but did notprevent the glutamate-induced mitochrondrialdepolarization and the secondary Ca2+ rise, but

they did exert protective effects against glutamate-induced neuronal death at 24 h following a 10 minexcitotoxic event. This protective effect againstglutamate-induced neuronal death apparently oc-curred through steps downstream of a sustainedincrease in Ca2+ associated with the collapse ofmitochondria potential.

Conclusion

Glutamate (MSG) causes motility abnormalitiesincluding low pulse numbers and swimming ces-sation in Aurelia ephyrae. Swimming and pulsingare impaired in a significant number of ephyraewithin 15 min of MSG treatment. The mechanismof MSG actions on ephyra motility is unknown,but glutamate is known to damage neurons andhair cells of higher animals possible through theformation of ROS. Microscopic fluorometricanalyses of calcium and free radicals, includingNO, indicated an increase of these substances inthe ephyrae as early as 5 min following MSGtreatment. Tests using DD-methionine and goldchloride, known antioxidants, revealed that bothcompounds accelerated the time of recovery fromMSG-induced motility impairment, possiblythrough their antioxidant activities.

Acknowledgments

Financial support for this research was providedby the Meridian Institute, Virginia Beach, VA, andby an Eastern Virginia Medical School Institu-tional Research Grant. We are also grateful for thetechnical assistance of Chris Maese.

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