PPNDS is an agonist, not an antagonist, for the ATP receptor of Paramecium
Dept of Biological Sciences, State University of NY at Buffalo, Buffalo, NY 14260, USA
* Author for correspondence (e-mail: thennes{at}acsu.buffalo.edu)
Accepted 28 October 2002
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Summary |
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Key words: PPNDS, ATP receptor, P2X1 antagonist, adaptation, desensitization, Paramecium, ß,-methylene ATP
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Introduction |
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In Paramecium, there are many types of depolarizing stimuli that
elicit graded, somatic depolarizations (receptor potentials) and consequent
Ca2+-based action potentials
(Eckert, 1972). These stimuli
include ionic stimuli (Machemer,
1988a
,b
),
anterior mechanosensory stimuli (Ogura and
Machemer, 1980
), heat
(Hennessey et al., 1983
) and
chemorepellents (Francis and Hennessey,
1995
) such as oxidants
(Hennessey et al., 1994
), GTP
(Clark et al., 1992
),
polycations (Hennessey et al.,
1995
) and ATP (Kim et al.,
1999
). These depolarizing receptor potentials are initiated on the
body (somatic) membrane and are conducted passively to the voltage-dependent
ciliary Ca2+ channels for action potential generation
(Eckert, 1972
; Machemer,
1988a
,b
).
When an action potential is produced, the Ca2+ influx causes
elevation of intraciliary Ca2+ and Ca2+-dependent
ciliary reversal. Therefore, a sufficiently strong depolarizing stimulus will
cause ciliary reversal and backward swimming. Some depolarizing stimuli (such
as Ba2+, Na+, lysozyme, oxidants and ATP) can cause
trains of action potentials and repetitive bouts of backward and forward
swimming called `avoiding reactions' (AR;
Jennings, 1976
). These
chemicals act as chemorepellents because the avoiding reactions re-orient the
swimming direction and result in movement away from the depolarizing stimulus
in a manner very similar to the `tumble' response of motile bacteria to
chemorepellents (Adler, 1987
;
Tso and Adler, 1974
;
Koshland, 1988
). The avoiding
reactions are easily observed under a simple dissecting microscope and serve
as the basis for many behavioral bioassays.
We have previously described ATP-induced AR, depolarizing receptor
potentials, external [32P]ATP binding, chemorepulsion and
chemosensory adaptation in the ciliate Tetrahymena
(Kim et al., 1999). We define
chemosensory adaptation as a decrease in responsiveness to a ligand as a
function of time of exposure to an appropriate concentration of that ligand.
This is different from the `adaptation', `accommodation' or `acclimatization'
responses previously described in Paramecium because they involve
long-term exposure to a high K+ stimulus and testing in a different
stimulus such as Ba2+, Mg2+ or heat
(Schusterman et al.,
1978
).
Chemosensory adaptation has been shown in responses to 15 min exposure to
micromolar quantities of GTP, ATP and polycationic chemorepellents [such as
lysozyme and PACAP (pituitary adenylate cyclase activating peptide)] in
Tetrahymena (Kuruvulla et al., 1997;
Kim et al., 1999;
Mace et al., 2000
), and
similar adaptation has been shown to lysozyme and GTP in Paramecium
(Kim et al., 1997
). Our
working model is that there are three separate chemorepellent receptors and
associated pathways (the GTP, `polycation' and ATP reception systems), because
cross-adaptation is not seen among the three systems. For example, adaptation
to 10 µmol l-1 GTP for 15 min causes a loss of GTP-induced AR,
GTP receptor potentials and surface [32P]GTP binding in
Paramecium without affecting [3H]lysozyme binding,
lysozyme receptor potentials and lysozyme-induced AR
(Kim et al., 1997
). In
Tetrahymena, no cross-adaptation was seen between the ligand binding,
behavior or receptor potentials associated with the responses to the three
representative chemorepellents GTP, ATP and lysozyme
(Kuruvilla et al., 1997
;
Kim et al., 1999
).
Cross-adaptation is seen when two ligands utilize the same receptor, causing
loss of responsiveness to both ligands following adaptation to one of them.
Such cross-adaptation has been shown in the responses to lysozyme and PACAP in
Tetrahymena, suggesting that they both activate the same `polycation
receptor' (Mace et al., 2000
)
or its adaptation pathway. Therefore, behavioral cross-adaptation can be used
as a first-screen bioassay to see whether two ligands may activate either the
same receptor or a common receptor adaptation pathway.
In vertebrates, ATP receptors are involved in neurotransmission, regulation
of blood flow and sensory transduction of signals for pain, tissue stretch and
temperature (Burnstock, 1996,
2000
;
Cook et al., 1997
;
Dubyak and El-Moatassim, 1993
;
Harden et al., 1995
;
Ralevic and Burnstock, 1998
).
Although ATP can be released from many types of cells, an extracellular ATP
signal often represents cell lysis and acts as a signal for cell and organ
damage and abnormal stretching or distention. It is this type of
cell-lysis-produced ATP that has recently been studied as a pain (nociceptive)
signal (Burnstock, 1996
;
Cook et al., 1997
;
McCleskey and Gold, 1999
;
Souslova et al., 2000
).
Response adaptation and receptor desensitization has been shown in several
types of ATP receptor systems (Burnstock,
2000
), and agonist-induced receptor internalization of native P2X1
has recently been demonstrated in a vertebrate preparation
(Ennion and Evans, 2001
). We
have proposed that ATP receptors in the ciliates Paramecium and
Tetrahymena are also acting as cell lysis detectors, providing a
general `blood in the water' danger signal so that these cells can avoid
whatever the situation was that caused nearby cell lysis. Therefore, the
ciliate ATP receptors are chemorepellent receptors.
There are many types of external purinergic receptors in vertebrates. In
general, responses to external purines are categorized as either P1 or P2,
with P1 referring primarily to responses to ligands such as adenosine while P2
ligands are generally nucleoside triphosphates such as ATP
(Abbracchio et al., 1993;
Khakh et al., 2001
). The P2X
receptors are also ion channels (ionotropic), while P2Y receptors are
G-protein coupled (metabotropic) (Khakh et
al., 2001
). There are currently seven different P2X receptor genes
cloned, and each of these subtypes of receptors (P2X1-P2X7) has indicative
agonist and antagonist specificities
(Khakh et al., 2001
). The
non-hydrolyzable ATP analog ß,
-methylene ATP is often used as the
preferred agonist in ATP receptor studies because ecto-ATPases and other
extracellular hydrolases are often present to hydrolyze external ATP. It has
been suggested that ecto-ATPases may be involved in the deactivation of
purinergic agonists in much the same way as acetylcholinesterase inactivates
acetylcholine signals (Kennedy et al.,
1997
; Hennessey et al.,
1997
). While ATP is a potent agonist for P2X receptors, the
non-hydrolyzable analogs, such as ß,
-methylene ATP, are often much
more potent (Burnstock, 2000
;
Khakh et al., 2001
).
To determine whether the properties of the ATP receptors of
Paramecium and Tetrahymena are similar to other known ATP
receptors, we followed a classical pharmacological approach to see if these
receptors fit into any of the known ATP receptor classifications. As the drug
pyridoxal-phosphate naphthylazo-nitro-disulfate (PPNDS) has been described as
a specific P2X1 receptor antagonist
(Lambrecht et al., 2000), we
tested it to see if it would block the responses of Paramecium to
external ATP and its analogs. Surprisingly, PPNDS produced the same
depolarizing responses as ß,
-methylene ATP, suggesting that PPNDS
is not an ATP receptor antagonist in Paramecium but an agonist. We
propose that the ability of PPNDS to cause inhibition of ATP responses is due,
at least in Paramecium, to time-dependent, agonist-induced ATP
receptor adaptation.
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Materials and methods |
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Chemicals and solutions
The `Paramecium wash buffer' solution contained 1.0 mmol
l-1 CaCl2, 1.0 mmol l-1 MOPS, 0.5 mmol
l-1 MgCl2, pH 7.2 with Tris-base. As PPNDS is a
tetrasodium salt, cells were preincubated for 15 min in the wash buffer
solution with the same Na+ concentration before exposure to PPNDS
to adapt them to the Na+. Similar Na+ balancing was done
with ß,-methylene ATP because it is a disodium salt. The solution
used for the in vivo [32P]ATP binding assays contained 10
mmol l-1 Tris-base, 0.5 mmol l-1 MOPS, 1.0 mmol
l-1 tartrate, 10 µmol l-1 EGTA, pH 7.2. PPNDS was
obtained from Tocris Cookson Inc., Ellisville, MO, USA, but all other
chemicals were purchased from Sigma-Aldrich, St Louis, MO, USA.
Behavioral assays
The chemorepellent behavioral assay used was the same as previously
described (Kim et al., 1997).
In this assay, individual cells were manually transferred to a test solution
using a micropipet and observed under the dissecting microscope for swimming
behavior. Cells showing any significant backward swimming events (`avoiding
reactions') during the first 5 s after transfer were scored as positive
responders. The avoiding reaction is defined by at least one backward movement
of at least one body length. Ten cells were observed for each trial, and the
mean ± S.D. was calculated for three trials (30 cells total) and
expressed as the percentage of cells showing avoiding reactions (% AR).
In the adaptation experiments (as in all experiments), cells were first
pre-incubated in the appropriate wash solution for 15-30 min before testing.
Cells were then placed in either 25 µmol l-1
ß,-methylene ATP (a non-hydrolyzable analog of ATP) or 100 µmol
l-1 PPNDS for varying amounts of time. Cells were transferred to
the same solution without the repellent for 20 s (as a brief wash) and then
transferred into the original test solution or a new test solution to assay
for repellent responses.
Electrophysiology
One-electrode intracellular membrane potential measurement procedures were
similar to those described previously (Kim
et al., 1997). The electrophysiological recordings were taken in
the behavioral solutions detailed above with 500 mmol l-1 KCl
electrodes (resistances of 100-200 m
). The membrane potentials were
recorded under continuous perfusion conditions at a rate of 15-20 ml
min-1, and bath volume was approximately 1 ml. In the adaptation
experiments, a cell was impaled in the repellent solution and exposed to
repellent perfusion for varying times. After a 15-20 s buffer perfusion wash,
the cell was re-exposed to the repellent perfusion.
In vivo binding assays
The in vivo [32P]ATP binding assays were performed in a
solution without added Ca2+ to prevent ATP hydrolysis by the
Ca2+-dependent ecto-ATPase
(Smith et al., 1997;
Hennessey et al., 1997
). In
the [32P]ATP binding assay, 0.2 ml of packed cells were washed
three times by centrifugation (700 g for 1 min) in the
solution and then 100 µl aliquots were withdrawn for binding assays. Each
100 µl aliquot was mixed with [32P]ATP and binding solution to a
final volume of 200 µl. This was vortexed gently and two 20 µl samples
removed and added to 2.5 ml of Ecoscint scintillation fluid for scintillation
counting. The average of these readings represented the sum of the bound and
free [32P]ATP. The remaining sample was pelleted by centrifugation
(500 g for 1 min) and 20 µl of the supernatant was removed
for scintillation counting. This represented the amount of free
[32P]ATP. The amount bound was determined by subtraction because
[bound] = [bound + free] - [free]. In the binding assay involving PPNDS, 0.2
ml of packed cells were washed three times by centrifugation in this solution
and diluted to a final volume of 2.0 ml. After a 15 min incubation in this
binding solution, PPNDS was added to a concentration of 100 µmol
l-1. Cells were incubated in 100 µmol l-1 PPNDS for
various times, and 100 µl aliquots were withdrawn and quickly washed by
centrifugation in 1 ml of binding solution. The resulting cell pellet was
vortexed with [32P]ATP and binding solution. The resulting mixture
was pelleted by centrifugation (500 g for 1 min). This pellet
was extracted and washed via centrifugation in binding solution then
placed into 2.5 ml Ecoscint for scintillation counting. This reading
represented the amount of [32P]ATP bound to cells. In the
competitive inhibition studies, [32P]ATP binding was performed in
the presence of a 50-fold concentration excess (compared with the
concentration of [32P]ATP present) of either cold (unlabelled)
ß,
-methylene ATP or PPNDS. This binding mixture was pelleted by
centrifugation (500 g for 1 min) and the pellet was extracted
and placed into 2.5 ml Ecoscint for scintillation counting.
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Results |
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Paramecium showed saturable external [32P]ATP binding in the in vivo binding assays (Fig. 3A). A Scatchard plot analysis of amount bound and free (unbound) [32P]ATP suggested a single set of external binding sites within the concentration range of 5-30 nmol l-1 (Fig. 3B). The apparent Kd was 23.1 nmol l-1, and the Bmax was 112 pmol l-1. Taking into consideration the number of cells in the assay, the number of functional surface receptors was estimated to be approximately 7.023x105 receptors cell-1.
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Continued exposure of cells to 25 µmol l-1
ß,-methylene ATP for 15 min resulted in a time-dependent
behavioral adaptation (Fig.
4A). This type of long-term adaptation was seen by exposing cells
to 25 µmol l-1 ß,
-methylene ATP for varying amounts
of time, transferring the cells to the wash buffer for 20 s and then retesting
the cells in 25 µmol l-1 ß,
-methylene ATP. This is
different from the short-term adaptation that was seen when cells were
observed continuously after transfer to 25 µmol l-1
ß,
-methylene ATP for several minutes (C. R. Wood and T. M.
Hennessey, manuscript in preparation). Short-term adaptation was seen as a
decrease in the frequency of AR within the first 10-30 s after transfer to
ß,
-methylene ATP and it roughly correlated with the duration of
the receptor potential. For example, a cell transferred to 25 µmol
l-1 ß,
-methylene ATP would show immediate, repetitive
AR but the frequency of AR decreased over time until, after approximately 30
s, the cell was swimming forward most of the time with very few AR. This is
short-term adaptation. However, if this cell (which had been exposed to 25
µmol l-1 ß,
-methylene ATP for 30 s) was transferred
to wash buffer for 20 s and re-exposed to 25 µmol l-1
ß,
-methylene ATP, it would show AR because it had not been in
ß,
-methylene ATP long enough to initiate long-term adaptation.
Electrophysiological analysis confirmed that receptor potentials could still
be generated upon re-exposure to 25 µmol l-1
ß,
-methylene ATP even after 8 min of adaptation but the amplitudes
were significantly decreased (Fig.
4B). Exposing cells to 25 µmol l-1
ß,
-methylene ATP for 15-20 min caused almost a complete loss of
both AR (Fig. 4A) and the
receptor potential (Fig. 4B),
defining conditions for long-term adaptation.
|
In summary, 25 µmoll-1 ß,-methylene ATP can
elicit a reliable, transient receptor potential that is the basis for the
ATP-induced AR. Long-term behavioral and electrophysiological adaptation is
complete after 15-20 min of exposure to 25 µmoll-1
ß,
-methylene ATP.
As there are many types of ATP receptors and they are often characterized
by their sensitivities to agonists and antagonists, we decided to see whether
a specific P2X1 inhibitor, PPNDS
(Lambrecht et al., 2000),
could eliminate the ATP responses of Paramecium. Instead of
inhibiting the type of responses we were assaying, PPNDS caused the same
responses as ß,
-methylene ATP. PPNDS produced the same kind of AR
as ß,
-methylene ATP in the concentration range commonly used in
the literature for inhibition of vertebrate P2X1 responses
(Lambrecht et al., 2000
). The
EC50 for PPNDS was approximately 70 µmoll-1, and
maximal responses were seen at approximately 120 µmoll-1
(Fig. 5A). Addition of 100
µmoll-1 PPNDS to ß,
-methylene-ATP-containing
solutions did not produce any immediate inhibition of AR because PPNDS itself
produced the same AR as ß,
-methylene ATP.
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PPNDS also produced similar transient depolarizations
(Fig. 5B) with similar
amplitudes and durations to those seen with ß,-methylene ATP. The
`receptor potentials' induced by 100 µmoll-1 PPNDS had a mean
amplitude of 13.2±2.2 mV and a mean duration of 29.2±7.3 s.
Using a standard t-test, there was no significant difference between
the amplitudes (t=0.46, P>0.1, d.f.=4) or the durations
(t=0.38, P>0.1, d.f.=4) of the ß,
-methylene
ATP-induced depolarizations and the PPNDS-induced depolarizations. Exposure of
cells to PPNDS for 15-20 min produced longterm adaptation as represented by a
time-dependent loss of both the PPNDS receptor potential
(Fig. 5C) and the PPNDS AR
(Fig. 6B). Therefore, the
behavioral and electophysiological responses to PPNDS are similar to the
responses to ß,
-methylene ATP and they both show the same type of
long-term adaptation.
|
Behavioral cross-adaptation was observed for ß,-methylene ATP
and PPNDS. Cells that were behaviorally adapted to 25 µmoll-1
ß,
-methylene ATP for 15 min lost AR after retesting in either 100
µmoll-1 PPNDS or 25 µmoll-1
ß,
-methylene ATP (Fig.
6A). Similarly, cells that were behaviorally adapted to 100
µmoll-1 PPNDS lost AR after retesting in either 100
µmoll-1 PPNDS or 25 µmoll-1
ß,
-methylene ATP (Fig.
6B). By contrast, cells that were behaviorally adapted to
ß,
-methylene ATP or PPNDS still retained full responsiveness to
another non-toxic, depolarizing chemorepellent, 10 µmoll-1 GTP
(Clark et al., 1992
). These
adapted cells also showed normal AR in the standard ionic depolarizing test
solutions (Saimi and Kung,
1987
) such as 40 mmoll-1 K+, 10
mmoll-1 Na+ and 8 mmoll-1 Ba2+.
This suggests that the adaptation is specific for the ATP-sensing pathway in
these cells and is not due to an overall desensitization.
Behavioral adaptation to 100 µmoll-1 PPNDS was associated
with a time-dependent decrease in external [32P]ATP binding sites
(Fig. 7A). Adaptation to 25
µmoll-1 ß,-methylene ATP for 15 min caused a similar
loss of external [32P]ATP binding
(Fig. 7B). This showed that
adaptation to either ß,
-methylene or PPNDS caused the loss of
external [32P]ATP binding. These changes in [32P]ATP
binding are not permanent but reversible. Cells were adapted to 100
µmoll-1 PPNDS for 15 min and were then transferred to the same
solution without PPNDS for an additional 15 min. These `de-adapted' cells
regained nearly 100% [32P]ATP binding, as did cells that were
previously adapted to ß,
-methylene ATP for 15 min and then washed
free of ß,
-methylene ATP and `de-adapted'
(Fig. 7B). Similar
reversibility (`de-adaptation') was seen in the behavioral and
electrophysiological changes described when adapted cells were incubated in
the ligand-free wash buffer for 15 min. We therefore do not consider this
time-dependent, PPNDS-induced loss of [32P]ATP binding to be true
antagonist-type inhibition because the well-known ATP receptor agonist
ß,
-methylene ATP has the same effect. Both
ß,
-methylene ATP and PPNDS are agonists in this system and
prolonged (15 min) exposure to either ligand causes the same behavioral
adaptation for the same reasons.
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If both ß,-methylene ATP and PPNDS activate the same receptor,
they may bind to the same active site. To test this hypothesis, we added a
50-fold excess of PPNDS to the in vivo [32P]ATP binding
mixture and immediately measured the amount of [32P]ATP bound.
Excess PPNDS caused an immediate decrease in [32P]ATP binding to
whole cells (Fig. 8),
suggesting that PPNDS may compete with [32P]ATP for binding to the
ATP receptor. Similarly, cold (non-radioactive) ß,
-methylene ATP
caused immediate inhibition of [32P]ATP binding when added in a
50-fold excess (Fig. 8). These
observations are consistent with the idea that ß,
-methylene ATP
and PPNDS may compete for [32P]ATP binding to the active site of
the ATP receptor; however, future purification of the ATP receptor is
necessary to confirm such competitive inhibition. In this respect, both
ß,
-methylene ATP and PPNDS are inhibitors of [32P]ATP
binding when they are present in the in vivo [32P]ATP
assay, but it must be remembered that cells were washed free of external
ß,
-methylene ATP and PPNDS in all previous experiments where
long-term, time-dependent adaptation was seen (see Figs
4,5,6,7,8).
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Discussion |
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Using Paramecium, we have shown that long-term behavioral
adaptation to ß,-methylene ATP occurs over a time span of
approximately 15 min (Fig. 4).
While the initial response of a cell to 25 µmol l-1
ß,
-methylene ATP is a depolarizing receptor potential (and
consequent AR), cells adapted for 15 min in 25 µmol l-1
ß,
-methylene ATP swim forward and show no ß,
-methylene
ATP-induced receptor potentials, even after a 20s wash and retest. As with
Tetrahymena (Kim et al.,
1999
), behavioral adaptation to ß,
-methylene ATP in
Paramecium is correlated with a loss in extracellular
[32P]ATP binding (Fig.
7). The loss of [32P]ATP binding prevents the
generation of the ß,
-methylene ATP-induced receptor potential, and
the result is no ß,
-methylene ATP-induced AR. We conclude that
Paramecium offer a unique opportunity to use quick, easy and humane
behavioral bioassays to screen the effects of purinergic agonists and
antagonists and then follow these behavioral screens with further
electrophysiological and [32P]ATP binding analysis.
The current literature suggests that one of the most specific anti-P2X1
antagonists to use for such a purpose is PPNDS
(Lambrecht et al., 2000), a
derivative of PPADS (pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid) with
reportedly higher specificity and potency than PPADS
(Khakh et al., 2001
). It has
been common in such studies to also expose a cell to a variety of agonists and
antagonists and to categorize the receptor on the basis of the spectrum of
sensitivities to these ligands (Khakh et
al., 2001
). If PPNDS is an antagonist of the ATP receptor of
Paramecium, it should inhibit ß,
-methylene ATP-induced
AR.
PPNDS, like ß,-methylene ATP, is an effective agonist for the
ATP responses of Paramecium. PPNDS causes the same depolarizing
responses as ß,
-methylene ATP. The behavioral EC50 is
higher for PPNDS than for ß,
-methylene ATP and it requires >100
µmol l-1 PPNDS for maximal stimulation. This is the approximate
PPNDS concentration used in the previous inhibition studies
(Lambrecht et al., 2000
).
PPNDS at 100 µmol l-1 produces transient, depolarizing receptor
potentials (Fig. 5B) that are
indistinguishable from those elicited by 25 µmol l-1
ß,
-methylene ATP (Fig.
2) in both amplitude and duration.
Prolonged (15 min) exposure to 100 µmol l-1 PPNDS produces
chemosensory adaptation, seen as a loss of PPNDS-induced AR and PPNDS receptor
potentials. Similarly, adaptation to ß,-methylene ATP causes
adaptation and loss of ß,
-methylene ATP-induced AR and
ß,
-methylene ATP receptor potentials. This type of loss in
responsiveness has also sometimes been called desensitization or
downregulation in the sensory literature but we chose the term adaptation to
encompass the behavioral, electrophysiological and receptor binding events.
The ATP receptor in rat vas deferens, which is a P2X1-like receptor, undergoes
similar agonist-induced desensitization
(Ennion and Evans, 2001
).
Cross-adaptation is seen between PPNDS and ß,-methylene ATP
responses, suggesting that they may be activating the same receptor. Cells
that have been adapted to 100 µmol l-1 PPNDS also lose
responsiveness to ß,
-methylene ATP (and vice versa). The
time courses of losses in ß,
-methylene ATP-induced AR are similar
for both PPNDS and ß,
-methylene ATP adaptation
(Fig. 6A). The opposite is also
true (Fig. 6B). Adaptation to
either PPNDS or ß,
-methylene ATP causes loss of external
[32P]ATP binding (Fig.
7A), which is reversible by a process we call `de-adaptation'
(Fig. 7B). Therefore, prolonged
(15 min) exposure to 100 µmol l-1 PPNDS causes a loss of
responsiveness to ß,
-methylene ATP because of cross-adaptation of
these responses. This is similar to the cross-adaptation we have seen between
ATP and ß,
-methylene ATP in both Paramecium (C. R. Wood
and T. M. Hennessey, unpublished observations) and Tetrahymena
(Kim et al., 1999
). This
adaptation was specific for ATP and PPNDS responses because we did not see any
cross-adaptation between PPNDS and GTP responses or ß,
-methylene
ATP and GTP responses in either Tetrahymena
(Kim et al., 1999
) or the
present study. Paramecium that were adapted to either PPNDS or
ß,
-methylene ATP were fully responsive to the other
chemorepellents GTP and lysozyme as well as to ionic stimuli (data not shown).
This is consistent with our previous hypothesis that there are at least three
separate chemorepellent pathways in Paramecium and each pathway shows
independent adaptation without affecting the sensitivities of the other two
pathways. We further proposed (Kim et al.,
1997
; Kuruvilla et al.,
1997
) that behavioral cross-adaptation is a convenient way to test
whether different ligands may activate the same receptor (or receptor
pathway).
If two ligands activate the same receptor by binding to the same site, they
should also compete for binding. We have shown that a 50-fold excess of either
ß,-methylene ATP or PPNDS can virtually eliminate all external
[32P]ATP binding in our in vivo assay
(Fig. 8) but a 50-fold excess
of GTP will not. These results support the hypothesis that both PPNDS and
ß,
-methylene ATP are agonists that share a common binding site on
the ATP receptor of Paramecium but GTP does not. The question of
whether PPNDS is a true competitive inhibitor for ATP binding to the ATP
receptor will be best approached when the ATP receptor of Paramecium
is purified for in vitro binding assays.
Preliminary observations in low Ca2+ solutions suggest the
EC50 for ATP to be approximately 200 µmoll-1 (S.
Zdep, personal communication), which is much higher than the measured
Kd value for [32P]ATP. This discrepancy may be
due to the fact that Ca2+ is included in the behavioral assays
(because it is necessary to show AR) but not in the binding assays.
Ca2+ can activate the Ca2+-dependent ecto-ATPase and
raise the EC50 because of ATP hydrolysis
(Hennessey et al., 1997). For
this reason, Ca2+ is omitted from the binding assays to inhibit
this ecto-ATPase (which has no known inhibitor in Paramecium).
However, such a large discrepancy between the EC50 and
Kd values for ATP suggests that the binding data may not
always fit quantitatively with the behavioral data for other reasons that we
have yet to resolve.
We conclude that PPNDS is an effective ATP receptor agonist (activator) in
Paramecium and it is not an antagonist in this system. PPNDS causes
the same depolarizing receptor potentials and avoiding reactions as the
well-known ATP receptor agonist ß,-methylene ATP, and
cross-adaptation is seen between these two agonists. The in vivo
[32P]ATP binding results are consistent with the hypothesis that
PPNDS and ß,
-methylene ATP both compete for the same ATP binding
site on the ATP receptor.
As the literature describes PPNDS as an antagonist in vertebrate cells, it
is possible that PPNDS only has agonist activity in Paramecium
because of a difference in the structure of the ciliate ATP receptor.
Considering the long evolutionary distance between the ciliates and
vertebrates, such a difference in agonist specificity cannot be ruled out. If
so, this ciliate ATP receptor may represent a new classification of ATP
receptors. This class would be easily recognized by the fact that PPNDS acts
as an agonist instead of an antagonist. However, it is possible that PPNDS may
also be an agonist in other systems where preincubation with 100
µmoll-1 PPNDS for 15 min is necessary for inhibition of an
ATP-dependent response. In some of these cases, it is possible that the loss
of ATP responses seen after prolonged exposure to PPNDS may be due to the same
agonist-induced adaptation (or `desensitization') that might be caused by any
agonist for this receptor (such as ß,
-methylene ATP) rather than
by the action of a true antagonist.
The distinction between ATP receptor antagonism and agonist-induced
adaptation is not a trivial matter when considering the mechanism of action of
new, experimental drugs. An example is the type of vertebrate ATP receptor
that is a specific type of pain receptor
(Cook et al., 1997). For these
receptors, ATP released from lysed cells causes pain by activation of ATP
receptors on specialized nerve endings. This type of preparation may prove
valuable for screening drugs that may act as local analgesics by blocking the
ATP receptors. However, it would be important to know that the drug does not
cause the pain that you want to prevent. Although sensory adaptation could
eventually result in analgesia by prolonged exposure to an agonist, the action
of a true antagonist might be preferred.
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