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Department of Biology, University of Vermont, Burlington, VT 05405
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • glutamate receptors • paramecium • protein kinase A • glutamate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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We have investigated the signal transduction pathway for glutamate in Paramecium tetraurelia by characterizing the behavior of cells, specific binding sites for glutamate and second messengers. Glutamate chemoresponse in P. tetraurelia has some attributes similar to the umami taste of monosodium glutamate and also some significant differences as described below.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Paramecium tetraurelia strain 51-S (sensitive to killer)
were grown as described in Sasner and Van Houten (1989)
.
Behavioral assays.
T-maze assays of chemoresponse were conducted as described in
Van Houten et al. (1982)
.
Cyclic nucleotide assays.
Assays for cAMP and cGMP were done using kits from Amersham
(Piscataway, NJ), as described in Yang et al. (1997)
.
Membrane potential measurements.
Measurement of membrane potential was done as in Preston and Van Houten (1987)
.
Binding studies.
Binding of 3H-glutamate to whole cells and cilia was
carried out as described in Yang (1995)
and as for
biotin and cAMP extracellular binding measurements (Bell et al. 1998
, Smith et al. 1987
).
Inhibitor studies in behavioral assays.
Cells were treated with H7 or H8 before T-maze assays of attraction
to glutamate and other stimuli as described in Yang et al. (1997)
.
Chemicals.
All chemicals were from Sigma (St. Louis. MO) or Calbiochem (San Diego, CA) unless otherwise indicated.
Affinity chromatography.
Glutamate agarose beads (Sigma) were used for affinity chromatography
generally as described for cAMP affinity chromatography (Van Houten et al. 1991
). Cell surface proteins were harvested in
salt/ethanol washes of cells (Capdeville et al. 1993
, Preer et al. 1981
) or Triton X100
detergent extracts of cell body membranes [pellicles, harvested as
described in Bilinski et al. (1981)
] and solubilized as
described in Van Houten et al. (1991)
.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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). To characterize the chemoreceptors for glutamate
attraction, we studied binding of 3H-glutamate to
whole cells by measuring displacement of
3H-glutamate from whole cells by unlabeled
glutamate or other unlabeled analogs. These studies revealed at least
two low affinity binding sites with similar
Kd of ~40 µmol/L
(Yang 1995
). In general, agreement was found between the
half-maximal response for behavior and the
Kd for binding of attractant stimuli
(Smith et al. 1987
, for example) with the
half-maximal value for behavior somewhat higher, indicating that
most binding sites must be occupied before a behavioral response is
elicited. However, for glutamate, the half-maximal values for
behavior and binding were different (1 mmol/L and 40
µmol/L, respectively). Although we cannot explain the
differences at this time, the studies of interference in binding and
behavior described below give confidence that the binding that we are
measuring is the relevant binding for chemoresponse.
Because inosine monophosphate
(IMP)4
and other 5'-ribonucleotides act synergistically with glutamate in
umami taste responses (Ugawa and Kurihara 1994
), we
tested for displacement of glutamate binding by these compounds. IMP
displaced glutamate from about half of the Paramecium
binding sites, but not from all of the specific sites (Fig. 1A
).Interestingly, quisqualate also displaced glutamate from some, but not
all specific sites (Fig. 1B
), and the combination of IMP and
quisqualate was as effective as glutamate in displacing all of the
specific 3H-glutamate binding (Fig. 1C
). Thus, IMP and quisqualate helped us to distinguish
between two glutamate binding sites.
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). However, glutamate completely interfered with chemoresponse to IMP,
which surprisingly is a repellent (Fig. 2B
). Quisqualate
interfered with glutamate attraction, even at very low ratios of
quisqualate to glutamate (data not shown; Yang 1995
).
Therefore, considering the results of the binding studies with these
behavioral studies, we believe that quisqualate, unlike IMP, could be
interfering with glutamate chemoresponse by interfering with binding of
glutamate to the chemoreceptor sites that mediate attraction to
glutamate.
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). However, GMP does not displace glutamate from whole-cell binding
sites and does not improve on the displacement of glutamate by IMP in
binding studies (Yang 1995
, data not shown). The
mechanism of this synergy is not yet known.
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Cells in glutamate swim smoothly and quickly, which is indicative of a
relatively hyperpolarized membrane potential; cells in IMP show
opposite behaviors, implying that the cells are relatively depolarized.
Hyperpolarization and depolarization and their concomitant effects on
ciliary beat and swimming patterns are associated with attraction and
repulsion, respectively (Van Houten 1994
). Direct
measurements of membrane potential of cells in glutamate relative to
cells in control buffers confirm that cells hyperpolarize ~8–10 mV
in 5 mmol/L K-L-glutamate relative to KCl (Yang 1995
).
The hyperpolarization is probably not due to the uptake of glutamate;
measurements are not made in Na+ salts but rather in
K+ salts, which inhibit glutamate uptake from the medium
(Yang 1995
). The mechanism of the hyperpolarization is
not yet known, but probably involves activation of a hyperpolarizing
K+ conductance initially to hyperpolarize the cell
(Preston and Usherwood 1988
). We do not know how the
glutamate receptor couples to the activation of a K+
channel. Interestingly, the hyperpolarization is sustained, and we
speculate that the stimulus (glutamate), bound to receptors, activates
adenylyl cyclase, which in turn activates protein kinase A (PKA) and
the calcium pump of the plasma membrane. Calcium pump activation could
then sustain the hyperpolarizing current (Fig. 4
). [See Van Houten (1994
and 1998)
for discussion.] The
inhibitory effects of the kinase inhibitors H7 and H8 on the glutamate
response support this possibility (Yang et al. 1997
).
Both of these inhibitors eliminate the attraction to glutamate but do
not affect attraction to other stimuli such as acetate and ammonium.
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, Hayashi et al. 1996
)
and also for metabotropic mGluR4 receptor in taste cells
(Bigiani et al. 1997
, Chaudhari et al. 1996
). Similarly, there is evidence for both mGluR1 and 3 in
catfish olfactory neurons (Medler et al. 1998
). The
pharmacology of the Paramecium glutamate receptor does
not seem to fit perfectly with these models (Yang 1995
).
However, the Paramecium glutamate response does share
some interesting characteristics with umami taste, specifically synergy
with a 5'-ribonucleotide, GMP. It may also share a sustained
hyperpolarization induced by glutamate in some rat taste cells
(Bigiani et al. 1997
). We have no evidence yet to
determine whether the Paramecium glutamate receptor
physically resembles the receptors implicated in taste (Bigiani et al. 1997
, Hayashi et al. 1996
) or in
olfactory neurons (Medler et al. 1998
). Considering that
the bacterial glutamate receptor in the periplasmic space shares
structural features with vertebrate metabotropic neurotransmitter
receptors, it is quite possible that the glutamate chemoresponse system
of Paramecium has retained these same features. The
answer to this awaits the purification of the P.
tetraurelia receptor. At present, we are using affinity
chromatography to identify proteins from the cell surface that bind to
glutamate (Bergeron, unpublished observations). There is a protein of
70 kDa that consistently elutes with glutamate from
glutamate-agarose columns.
Several species of bacteria use extracellular cues to determine the
density of their own populations and, thereby, judge the optimum time
at which to express a set of genes or a behavior (Swift et al. 1996
). A phenomenon called "quorum sensing" allows the
bacteria to respond in almost a step function only when conditions are
right, i.e., when some important cue is in adequate concentration. It
is not unusual for paramecia to respond only to relatively high (10
µmol/L-1 mmol/L) concentrations of stimulus in their
chemically noisy pond environments [see Van Houten (1994)
for discussion]. We consider the
Paramecium response at relatively high concentrations of
stimuli such as glutamate to be a form of quorum sensing—not sensing
of other paramecia but of their prey, bacteria, which are good sources
of glutamate. It does not benefit a paramecium to dash off through the
pond after a stray signaling molecule. It is more efficacious for the
paramecia to wait until the bacterial population has risen, producing
an elevation in glutamate concentrations to some threshold value before
responding with an attraction response. [See Bell et al. (1998)
and Van Houten (1994)
for discussion.]
It is tempting to speculate that the synergy between GMP and glutamate
enhances locating and foraging on actively growing bacteria by
enhancing sensitivity of chemoresponse, whereas the repellent signal
from IMP warns against toxic conditions. Perhaps understanding the use
of glutamate by Paramecium to accumulate near bacteria
will provide some insight into the nuances among the glutamate
receptors involved in sensory systems such as taste or olfaction in
aquatic organisms and the receptors used in neurotransmission.
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FOOTNOTES |
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2 Supported by National Institutes of Health DC
00721 and the VCC.
4 Abbreviations used: GMP, guanosine
monophosphate; IMP, iosine monophosphate; PKA, protein kinase A.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2.
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