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Toxicon Vol. 34, No. 4, pp. 443-450, 1996
0041-0101(95)00150-6
ANTAGONISM OF GLUTAMATE RECEPTOR BY A
CHROMATOGRAPHIC FRACTION FROM THE
EXUDATE OF SEA ANEMONE PHYLLACTIS
FLOSCULIFERA.
ANOLAND GARATEIX1, AMIRA FLORES2, c,
JUAN M. GARCÍA-ANDRADE3, ADYS PALMERO1,
ABEL ANEIROS1, ROSARIO VEGA3 and ENRIQUE SOTO3*.
1Centro de Bioactivos Marinos, Ministerio de Ciencia Tecnología y Medio Ambiente, la Habana,
Cuba. 2Facultad de Medicina and 3Instituto de Fisiología, Universidad Autónoma de Puebla,
Apartado Postal 406, Puebla, Pue. 72000, México.
(Received 18 September 1995; accepted 3 November 1995)
A. Garateix, A. Flores, J.M. García-Andrade, A. Palmero, A. Aneiros, R. Vega and E. Soto.
Antagonism of glutamate receptor by a chromatographic fraction from the exudate of sea anemone
Phyllactis flosculifera. Toxicon 34, 443-450, 1996. - In the search for new glutamate antagonists it
seems promising to characterize the effects of venom from invertebrates that prey mainly on
crustaceans. In this work, the exudate of the sea anemone P. flosculifera was used as a source of
this kind of compounds. The action of chromatographic fraction D from P. flosculifera was tested
upon microiontophoretically evoked glutamate responses in intracellular recordings from central
neurons of the land snail Zachrysia guanensis. Bath application of fraction D (2-8 mg/ml, n = 13)
diminished both the excitatory and the inhibitory components of glutamate agonists in Z. guanensis
neurons; this action was dose-dependent and partially reversible. Fraction D actions were also
tested in the multiunit spontaneous and mechanically evoked responses of the glutamatergic
junction between hair cells and afferent neurons of the axolotl Ambystoma tigrinum. Pressure
ejection of fraction D in concentrations ranging from 0.5 to 2 mg/ml (n = 9) decreased the
spontaneous and mechanically evoked activity of semicircular canal afferent neurons and the
responses evoked by kainic acid and α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid. This
action was also dose-dependent and partially reversible. These results indicate that fraction D acts
as a glutamate receptor antagonist in snail and amphibian neurons. Further studies are required to
characterize the active compounds responsible for this action and its specificity upon the subtypes
of glutamate receptors.
A. GARATEIX et al. 444
INTRODUCTION
Glutamate is the major excitatory transmitter in the mammalian Central Nervous System (CNS).
Besides their normal physiological role, glutamatergic receptors seem to play a significant role in
the origin of many pathological phenomena in the CNS. Thus, the finding of toxins in venoms of
certain wasps and spiders that antagonize glutamate responses in different biological models has
generated a great deal of interest (Usherwood et al., 1984; Usmanov et al., 1985; Akaike et al.,
1987; Early and Michaelis, 1987; Eldefrawi et al., 1988; Bruce et al., 1990) becaause of its potential
for clinical and research applications.
The development of glutamate receptor antagonists seems a promising strategy for finding new
tools for neurobiological research, and may eventually lead to the discovery of substances with
important therapeutic possibilities. One source of glutamatergic antagonists has been the venoms of
some species of spiders (Akaike et al., 1987; Jackson and Usherwood 1988; Kawai, 1991). These
low mol. wt toxins have been shown to block glutamatergic synapses in invertebrates and also in
the mammalian CNS (Akaike et al., 1987).
It has been clearly shown that glutamate is the excitatory neurotransmitter in crustacean
neuromuscular junction (Takeuchi and Takeuchi, 1964); thus, it seems promising to search for
glutamate receptor antagonists among the components of the venom from invertebrates that prey
mainly on crustaceans as is the case with sea anemones.
Despite reports of a high mol. wt substance with glutamate receptor antagonistic properties
being obtained from the pelagic coelenterate Physalia physalis (Mas et al., 1989), sea anemones
have not been previously used as a source of this type of compounds. In previous experiments we
have observed that the exudate of the sea anemone Phyllactis flosculifera diminishes the responses
to the microiontophoretical application of L-glutamate in molluscan neurons. The present work
reports results of studies of a chromatographic fraction (fraction D, containing compounds with
mol. wt under 1000 Da) obtained from the sea anemone P. flosculifera on the glutamate-mediated
responses of two biological preparations.
MATERIAL AND METHODS
Intact specimens of P. flosculifera were collected in the northern sea shore of Havana, Cuba. Immediately
after its capture, a set of one kilogram of anemones, were stressed by immersion in distilled water (500 ml) for
about 10 min. The tissue exudate obtained by this procedure was decanted, centrifuged, freeze dried and kept
at -20° C until use. It could not be excluded that osmotic shock will probably induce an exudate from epithelial
cells, mucous cells and cnidocytes from the epidermis of the anemone; however, after this treatment the
animals recovered and were kept in laboratory conditions for further “milking” procedures. The exudate was
gel filtered on Sephadex G-50 in the same way as previously described for other species of sea anemone
(Aneiros et al., 1993). Fraction D eluted approximately at one column volume, it contains low mol. wt (under
1000). This fraction was lyophilized and used in the electrophysiological experiments as dried weight. Further
purification of those chromatographic fractions with biological activity is now in progress.
Snail neuron experiments
A group of experiments was carried out on identified neurons of the land snail Zachrysia guanensis. The
suboesophageal ganglion was dissected according to the procedure described previously (Martínez Soler et al.,
1983). The preparation was kept in a "snail" solution with the following composition (mM): NaCl 80, KCl 4,
CaCl2 7, MgCl2 4, Tris HCl 10, pH adjusted to 7.4. Intracellular recordings were made using glass
microelectrodes filled with 3 M KCl and resistance from 10 to 20 MΩ. The indifferent electrode was a silver
plate covered with agar-KCl and immersed in the bathing solution. The recording microelectrode was
connected to a microelectrode pre-amplifier (MEZ 8201, Nihon Kohden). The signal was send to an
oscilloscope and digitized using an AD converter for further analysis.
Sea Anemone Glutamate Antagonists 445
For the iontophoretic ejection of L-glutamate, glass microelectrodes were filled with 1 M sodium
glutamate (Merck) with a resistance of about 100 MΩ. The compound was ejected using current pulses of 1.5
µA and 20-300 ms. Fraction D was applied by bath perfusion.
Once a successful intracellular recording was established, control responses to microintophoretic
application of glutamate were studied; thence, fraction D was bath-applied for up to 20 min time during which
responses to glutamate were tested every 2 min. The amplitude of the depolarization and inhibitory phase
induced by glutamate was measured and compared with control responses. Experiments were carried out at
room temperature. To construct the dose response relationship for fraction D the depolarization induced during
the excitatory phase was expressed as a mean percent of change with respect to control conditions ± S.E.M.
Isolated inner ear experiments
Experiments were also performed in the isolated inner ear of the axolotl (Ambystoma tigrinum). Briefly, larval
axolotls weighing 30 - 60 g were decapitated, and the otic capsule opened ventrally. The nerve fibers of the
anterior and lateral canals were dissected up to the brainstem. The cartilaginous otic capsule was cut and
isolated from the cranium. The isolated inner ear was transferred to a recording chamber and continuously
perfused with Ringer solution of the following composition (in mM): KCl 2.5, NaCl 111, CaCl2 1.8, glucose
10, HEPES 5, pH 7.4. (Soto and Vega, 1988; Soto et al., 1994). Extracellular multiunit recordings of the
semicircular canal afferent fibers were obtained using a suction electrode. The signal was amplified using an
AC amplifier (Grass P-15) and monitored in an oscilloscope (Tektronix, 5111). In some experiments
intracellular recordings of afferent neurons were performed by using quartz glass microelectrodes filled with
potassium acetate 3 M with resistance of 100 MΩ. In this last case signals were led to a DC amplifier
(Axoprobe 1A). In both types of recordings, the signal was also send to a magnetic tape recorder (Dagan,
Unitrade) and to a computer for its analysis in the form of frequency vs time plots and for the measurement of
the amplitude and frequency of EPSPs (Soto and Vega, 1987). In extracellular multiunit recording
experiments, the recording chamber, the amplifier and manipulators were mounted on a rotating table driven
by a DC servo-controlled motor (Aerotech, 49179). Sinusoidal accelerations (0.2 Hz) were induced by using a
function generator (Hewlett Packard, 8904A). The preparation was usually stimulated for 30 s periods under
control conditions and 30 sec, 1.5 min and 5 min after drug administration.
Drugs were applied by pressure ejection from a pipette (100 µm tip diameter) with a flow rate of 10 µl/s
for 2 s positioned in the vicinity (0.5 mm) of the origin of the afferent fibers at the ampullary level. The
concentrations given herein are those originally in the pipette. Since the bath volume was 2 ml, the drug
concentration in the ampullary vicinity decays exponentially to about 1% of its original value in a few
milliseconds. Kainic acid (KA), α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA), and
quisqualic acid (QA) were bought from RBI, N-methyl-D-aspartic acid (NMDA) from CRB.
To construct the concentration-response relationship, the spike discharge was normalized as a percent of
change with respect to control conditions. Comparison of the mechanical responses were done by obtaining the
mean of the peak response in at least three cycles of the sinusoidal stimulus period (the first and the last cycles
were eliminated).
RESULTS
Snail neurons
Microintophoretic application of glutamate in central neurons of Z. guanensis produces two types
of responses: 1) excitatory, characterized by a depolarizing phase with an increase of the spike
activity of the neuron and, 2) biphasic, composed by an excitatory component followed by a
hyperpolarizing phase of longer duration (Martínez Soler et al., 1983).
A. GARATEIX et al. 446
A preliminary characterization of the glutamate receptors present in Z. guanensis neurons was done
using the glutamate agonists: KA (1-5 mM, n = 7), QA (0.4-2 mM, n = 6) and NMDA (0.1-5 mM,
n = 6). Typically, the effects of microintophoretic applications of glutamate were diminished
Fig. 1. Effect of glutamate agonists bath perfusion on the response of Z. guanensis neurons to
microiontophoretic glutamate application.
Responses to microiontophoretically applied Glutamate (1 M, 1.5 µA, 200 ms) before (A) and after
(B) bath perfusion of kainic acid (KA, 5 mM), quisqualic acid (QA, 0.4 mM) or N-methyl-D-aspartic
acid (NMDA, 1 mM). The point indicates the application of glutamate.
Calibration: vertical bar = 50 mV, horizontal bar = 2 s.
Fig. 2. Effect of fraction D on a glutamate-induced response of a Z guanensis neuron.
Intracellular recordings of the response to microiontophoretically applied glutamate (1 M, 1.5 µA, 200 ms)
before (A) and 7 minutes after exposure (B) to fraction D (8 mg/ml). (C) Response to glutamate after 15
min washing recovers up to 60% of control value. The point indicates the application of glutamate.
Calibration: vertical bar = 50 mV, horizontal bar = 2 s. (D) Dose-response curve for fraction D on the
excitatory phase of the glutamate responses in Z. guanensis neurons. Circles represent the mean values of
2-4 experiments ± SE.
Sea Anemone Glutamate Antagonists 447
by the simultaneous bath perfusion of KA and QA, whereas the perfusion of NMDA did not cause
any change in the glutamate-evoked responses (Fig. 1).
The perfusion of fraction D decreased the glutamatergic response (2-8 mg/ml, n = 13), as
depicted in figure 2; its effect develops gradually depending on the concentration used, and its
action was dose-dependent and partially reversible in a range of about 30 min after washing the
preparation with normal snail solution. Since the inhibitory phase of the response was very variable
and masked by the spontaneous spike discharge of the neurons, only the effects on the excitatory
phase of glutamate action were measured (Fig. 2D).
Isolated inner ear
Pressure ejection of chromatographic fraction D (n = 6, 0.5-2 mg/ml) inhibited the basal and the
mechanically evoked electrical activity of the semicircular canal afferent fibers within the first 30
sec of application (Fig. 3). This action was dose-dependent and partially reversible after 15 min
washing with normal amphibian solution. Its inhibitory potency was greater upon the basal activity
with a CI50 of 0.5 mg/ml for the basal activity and a CI50 greater than 2.0 mg/ml for the
mechanically evoked responses (Fig. 3).
To determine whether fraction D acts at the presynaptic level on the hair cells (i.e. decreasing
the release of neurotransmitter), or postsynaptically at the afferent fibers level (i.e. blocking the
neurotransmitter receptors), we studied the capability of this substance to block competitively the
action of glutamate receptor agonists, and examined its action upon the EPSPs intracellularly
recorded in the afferent neurons. The responses to KA (30 µM, n = 4) and to AMPA (300 µM, n =
Fig. 3. Fraction D diminishes the basal and mechanical responses of the semicircular canal
afferent fibers.
(A) Frequency-time plots of the basal and mechanically evoked discharge of semicircular
canal afferent neurons. (B) After ejection of 20 µl, 2 mg/ml of fraction D. Gray bars indicate
the time during which the preparation is sinusoidally stimulated at 0.2 Hz. (C) Dose-response
relationship between fraction D concentration and semicircular canal afferent fibers basal and
mechanically evoked electrical activity. Each point represents the mean of at least three tests
of a given concentration ± SE.
T I M E (min) FRAC. D (mg/ml)
A. GARATEIX et al. 448
3) were studied in control conditions and after the application of fraction D (2 mg/ml).
Microperfusion of both KA and AMPA exerted a very strong excitatory action upon the afferent
fibers' spike discharge (Fig. 4). The excitatory action of KA and AMPA was reduced in a 31% and
22% respectively by previous (1 min) ejection of fraction D (Fig. 4). The time courses of KA and
AMPA effects were slightly prolonged by fraction D, which also decreased their characteristic
postexcitatory inhibition.
In the intracellular recordings from afferent neurons, bath perfusion of fraction D 1.5 mg/ml (n
= 3) diminished the spike discharge. Analysis of the number and amplitude of EPSPs recorded in
control conditions and after fraction D administration reveals that the toxin modifies the amplitude
of the EPSPs and also decreased their frequency, indicating that it exerts both presynaptic and
postsynaptic effects.
DISCUSSION
Both snail and axolotl experiments provide evidence indicating that fraction D components act as
glutamate receptor antagonists.
Since intracellular recording of electrical activity of snail neurons has been shown to be a very
reliable technique and their pharmacological properties are well known (Kerkut et al., 1975; Rózsa,
1984); we decided to use Z. guanensis neurons as a model to test the action of chromatographic
fractions extracted from sea anemones.
The action of KA and QA on the responses evoked by the microintophoretic application of
glutamate and the lack of effect of NMDA shows that Z. guanensis neurons possess somatic
Fig. 4. Fraction D reduced the excitatory effects of kainic acid (KA) and α- amino-3-hydroxy-5-
methylisoxasole-4-propionic acid (AMPA) upon the semicircular canal afferent fibers.
(A, B) Responses of semicircular canal afferent neurons to the ejection of KA (30 µM, 20 µl) in
control conditions and in the presence of fraction D (2 mg/ml) respectively. (C, D) Similar
experiment using AMPA (300 µM, 20 µl).
Sea Anemone Glutamate Antagonists 449
excitatory amino acid receptors of the non-NMDA type: NMDA receptors do not appear to be
expressed in these neurons, as had been previously reported for other molluscan neurons (Ascher et
al., 1986; Bolshakov et al., 1991).
The perfusion of fraction D obtained from the sea anemone P. flosculifera diminishes the
glutamatergic responses in Z. guanensis neurons. This indicates that this anemone contains
substances with affinity for the glutamatergic receptors, presumably of non-NMDA type.
Previous studies have shown that synaptic transmission between hair cells and afferent neurons
in the amphibian inner ear is mediated by an excitatory amino acid (Annoni et al, 1984; Soto and
Vega, 1988; Soto et al., 1994). It has been found that both NMDA and non-NMDA receptors
participate in the afferent fibers' response to the hair cell transmitter (Soto et al, 1994). Since
vestibular afferent neurons are readily accessible for recording and isolated inner ear preparations
allow for the study of natural (mechanical) and pharmacological stimulation, we selected this as an
additional model system to test the action of chromatographic fractions extracted from sea
anemones.
The dose-dependent inhibitory action of fraction D on the basal and the mechanically evoked
spike discharges of the vestibular afferent neurons support the idea that this fraction acts as a
glutamate receptor antagonist in amphibian neurons.
Fraction D also diminished the excitatory responses evoked by KA and AMPA on the
semicircular canal afferent neurons, indicating that it acts at the postsynaptic level, as an antagonist
of KA and AMPA receptors. The analysis of the frequency and amplitude of EPSPs recorded from
the afferent neurons also suggests a postsynaptic effect, but it does not allow us to role out a
presynaptic action, since a modification of the frequency of occurrence of EPSPs was also
produced.
As in the case of spider venoms (Early and Michaelis, 1987; Jackson and Usherwood, 1988),
glutamate has been shown to be a neurotransmitter in coelenterates; thus, their venom could contain
glutamate in their exudate. Glutamate concentrations equivalent to the quantity that may be
contained in fraction D had no effect on the snail neurons. Furthermore, fraction D induced no
excitatory action in the semicircular canal afferent neurons, as could be expected if some excitatory
amino acid receptor agonists were present.
A long polypeptide toxin has been purified from P. flosculifera (Kem et al., 1987).
Nevertheless, fraction D activity could not be explained on the basis of this toxin because it
contains compounds with mol. wts of less than 1000. Moreover, the reversibility of fraction D
effects in both preparations and their long duration after fraction D application indicate that no
cytotoxic action takes place in our experimental conditions.
In both Z. guanensis neurons and isolated inner ear, the possibility that some components of
fraction D interact with other types of receptors present in these cells can not be excluded,
particularly because cholinergic receptors have been shown to exist in both types of preparations
(Martínez-Soler et al., 1983; Rózsa, 1984).
Our results indicate that fraction "D" acts as a glutamate receptor antagonist in snail and
amphibian neurons. Further experiments will be performed to identify the component, or
components, responsible for the antiglutamatergic effect present in the exudate of this sea anemone,
and to determine its specificity upon the different subtypes of glutamate receptors.
Acknowledgments- The authors wish to thank Dr. Bjorn Holmgren for critical reading of the
manuscript and professor Augustine Udeh for proof reading the English manuscript. This work was
partially supported by CONACyT grant 2133-N to ES.
A. GARATEIX et al. 450
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