1Division of Neuroscience and 2Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research; and 3Division of Biochemistry and Molecular Biology, The Faculties, Australian National University, Canberra, ACT 0200, Australia
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ABSTRACT |
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de Plater, G. M.,
P. J. Milburn, and
R. L. Martin.
Venom From the Platypus, Ornithorhynchus anatinus,
Induces a Calcium-Dependent Current in Cultured Dorsal Root Ganglion
Cells.
J. Neurophysiol. 85: 1340-1345, 2001.
The platypus (Ornithorhynchus
anatinus), a uniquely Australian species, is one of the few
living venomous mammals. Although envenomation of humans by many
vertebrate and invertebrate species results in pain, this is often not
the principal symptom of envenomation. However, platypus envenomation
results in an immediate excruciating pain that develops into a very
long-lasting hyperalgesia. We have previously shown that the venom
contains a C-type natriuretic peptide that causes mast cell
degranulation, and this probably contributes to the development of the
painful response. Now we demonstrate that platypus venom has a potent
action on putative nociceptors. Application of the venom to small to
medium diameter dorsal root ganglion cells for 10 s resulted in an
inward current lasting several minutes when the venom was diluted in
buffer at pH 6.1 but not at pH 7.4. The venom itself has a pH of 6.3. The venom activated a current with a linear current-voltage
relationship between 100 and
25 mV and with a reversal potential of
11 mV. Ion substitution experiments indicate that the current is a
nonspecific cationic current. The response to the venom was blocked by
the membrane-permeant Ca2+-ATPase inhibitor,
thapsigargin, and by the tyrosine- and serine-kinase inhibitor, k252a.
Thus the response appears to be dependent on calcium release from
intracellular stores. The identity of the venom component(s) that is
responsible for the responses we have described is yet to be determined
but is probably not the C-type natriuretic peptide or the defensin-like
peptides that are present in the venom.
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INTRODUCTION |
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Many invertebrate and vertebrate
species produce venoms that are predominantly used either for food
procurement or in self-defense. In keeping with these functions, such
venoms contain peptides and other molecules that can induce
immobilization and death. By contrast, the venom of one of the few
living venomous mammals, the platypus (Ornithorhynchus
anatinus), is believed to be used in defense of breeding territory
(Grant and Temple-Smith 1998). Venom is produced in the
crural glands of the male during the breeding season and is
aggressively inflicted through a calcaneous spur on each hindlimb
(Grant 1995
). Because the venom appears to have a
different function from venoms produced by nonmammalian species, it may
contain peptides or molecules whose principal effects are non-life
threatening but nevertheless may seriously impair the victim.
That this could be the case is evident from the symptoms of platypus
envenomation. In the human the most remarkable symptom is an immediate
and excruciating pain (Fenner et al. 1992; Martin and Tidswell 1895
; Spicer 1876
; Tonkin
and Negrine 1994
). Edema rapidly develops around the wound and
gradually spreads throughout the affected limb (Fenner et al.
1992
; Martin and Tidswell 1895
; Spicer
1876
; Tonkin and Negrine 1994
). Information
obtained from case histories (Fenner et al. 1992
;
Tonkin and Negrine 1994
) and anecdotal evidence
indicates that the pain develops into a long-lasting hyperalgesia that
persists for days or even months. Morphine analgesia partially
alleviates the pain, but, in the one detailed case history, wrist block
was necessary to effect complete pain relief (Fenner et al.
1992
). These symptoms differ markedly from those associated with envenomation by snakes, for example, which can include systemic effects such as paralysis, myolosis, defibrination coagulopathy, and
renal failure.
Recently we commenced a physiological and biochemical characterization
of Ornithorhynchus anatinus venom (OaV). Like
other venoms, it contains an hyaluronidase, which probably facilitates the spread of the venom through tissues (de Plater et al.
1995). It also contains a C-type natriuretic peptide,
Ov-CNP (de Plater et al. 1995
), that results in substantial
edema when subcutaneously injected (de Plater et al.
1998b
). This probably reflects Ov-CNP-induced mast
cell degranulation (de Plater et al. 1998b
) and is
consistent with reduction, in the rat, of the edema by the 5-HT2
receptor antagonist, ketanserin (de Plater et al. 1995
).
The venom-induced mast cell degranulation suggests that some of the
pain results from release of inflammatory mediators. However, the venom
may also act on nociceptors themselves. To test this hypothesis, we
applied OaV to small-medium diameter (20-40 µm) cultured
dorsal root ganglion neurons (DRGs). These neurons are believed to
exhibit properties that reflect those of peripheral nociceptor
terminals in vivo (Gold et al. 1996; Senba and
Kashiba 1996
).
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METHODS |
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Cell preparation
Procedures used were very similar to those described by
Gold et al. (1996), in which the properties of DRG
neurons in vitro have been characterized. Thoracic and lumbar dorsal
root ganglia were obtained from 5- to 8-wk-old Wistar rats that were
decapitated under halothane anesthesia (4% in
O2). All experimental procedures were approved by
The Animal Experimentation Ethics Committee at the Australian National
University. Ganglia were dissociated at 36°C for 45 min in H-16
Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 units/ml penicillin G and 0.1 mg/ml streptomycin, and containing 2.5 U/ml collagenase (type I). After washing, the ganglia were incubated in
5 mg/ml dispase at 36°C for 30 min, then resuspended in DMEM
supplemented with 10% fetal calf serum and the cell bodies dissociated
by trituration. Cells were distributed in tissue culture plates
containing coverslips coated with collagen and
poly-D-lysine and incubated in CO2 at
37°C until used. For electrophysiological recording a coverslip was
placed in the bath on the stage of an inverted microscope, and the
cells were perfused at 1-2 ml/min with an HEPES-buffered solution of
the following composition (in mM): 130 NaCl, 3 KCl, 1.2 NaHCO3, 0.6 MgCl2, 2.5 CaCl2, 10 HEPES, and 10 glucose, titrated to pH
7.4 with NaOH and osmolarity adjusted to 320 mOsm with sorbitol.
Electrophysiology
Whole cell recordings were made at room temperature from 20-40
µm diameter DRGs between 6 and 24 h after plating; only cells that had smooth membranes under phase contrast microscopy were selected
for study. The recording pipettes contained (in mM) 100 KCl, 2 2Na-ATP,
0.5 2Na-GTP, 11 EGTA, 1 CaCl2, 2 MgCl2, and 10 HEPES, titrated to pH 7.2 with KOH
to give a final [K+] of 147 mM, and adjusted to
305 mOsm with sorbitol. Currents were measured using an Axopatch 1D
(Axon Instruments) and data acquired on an IBM-PC compatible computer
using pClamp software (20-kHz digitization rate). Slow records of the
experimental measurements were made using a MacLab (AD Instruments),
MacIntosh computer, and a digitization rate of 2 Hz. Electrode
resistance ranged from 1.5 to 3 M, series compensation from
80-90%, and corrections were applied for liquid junction potentials
(JPCalc, Prof. P. H. Barry, UNSW, Australia). Resting membrane
potential was measured prior to OaV or drug application, and
only cells with corrected resting membrane potentials more negative
than
50 mV were studied.
Materials
Whole venom from Ornithorhynchus anatinus was diluted at a concentration of 1 mg/ml in either MES-buffered solution at pH 6.1 (composition as for the HEPES-buffered solution but pH adjusted to 6.1 with HCl). The osmolarity of OaV-containing solutions was adjusted to 320 mOsm using sorbitol. The venom was applied to individual neurons by pressure ejection (5-10 psi) through flow pipes attached to fine plastic tubing that was back-filled with 100-300 µl of test solution. Other drugs used were capsaicin (Fluka Chemica), freshly prepared from stock to a final concentration of 1 µM in 0.01% ethanol, and thapsigargin (Research Biochemicals International) and k252a (Alomone Labs), each prepared from stock to give a final solution that contained 0.02% DMSO.
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RESULTS |
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Of 96 DRGs subjected to a 10-s application of 1 mg/ml of
OaV diluted in either MES or HEPES at pH 6.1, 69 responded
with a long-lasting (duration 246.7 ± 36.5 s, mean ± SE) inward current at 60 mV whose average latency to onset
was 23.3 ± 3.0 s. The current was characterized by multiple
transient events (Fig. 1A) and
had an estimated mean peak amplitude of
4.65 ± 0.34 nA. Neurons that failed to respond to OaV (n = 27)
usually responded to either buffer at pH 6.1 or to 1 µM capsaicin
with a single, transient inward current (Fig. 1B). Prior to
venom application the resting membrane potential averaged
59.8 ± 1.7 mV (n = 59) for responsive neurons and
60.9 ± 1.5 mV (n = 24) for the unresponsive
neurons. When OaV was buffered at pH 7.4, its application to
DRGs produced little or no response (n = 5, Fig.
1C). There was no correlation between sensitivity to
capsaicin and OaV (n = 10; resting membrane potential prior to OaV application
60.4 ± 2.5): of
six capsaicin-sensitive neurons, four responded to OaV and
of four capsaicin-insensitive neurons, three responded to
OaV.
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To determine the ionic basis of the OaV-induced current,
voltage ramps from 110 to 60 mV were applied at a rate of 226 mV/s (Fig. 2, A-D). Subtraction of
the curve obtained before OaV application from that obtained
during its application yielded a linear inward current-voltage
(I-V) relationship in the
100- to
50 mV range, which
suggests activation of a voltage-independent current. At values more
positive than
50 mV, another inward current was observed; but when
K+ channels were blocked (see below),
this current was not observed. Thus it is not likely to be a
venom-activated current and probably reflects changes in the quality in
the voltage-clamp between voltage ramps due to the large current
flowing across an incompletely compensated access resistance. From this
point on, we concentrated on characterizing the voltage-independent
current. The linear range (r = 0.97-0.99) of the
I-V relationship was extrapolated to give a mean
reversal potential for the OaV-activated current of
11.4 ± 2.0 mV (n = 22; Fig. 2C).
This is close to the calculated ECl of
6.9 mV and to Erev for the
nonselective cationic current that is known to occur in DRGs
(Crawford et al. 1997
; Currie and Scott
1992
), thus suggesting the involvement of either
Cl
and/or nonselective cationic currents.
Replacement of extracellular (perfusate) Cl
with gluconate caused a significant shift in
Erev to 5.2 ± 1.9 mV
(n = 4; P < 0.01), but this value fell
far short of the calculated ECl of
61.6 mV. Replacement of recording pipette Cl
with gluconate was without major effect (n = 5;
Erev of
1.1 ± 3.5 mV compared
with calculated ECl of
70.7 mV).
These data suggest that the OaV-induced current is
dominantly carried by cations rather than by
Cl
. We also studied the linear current when
pipette K+ was replaced with TEA to block a
variety of K+ currents (Hille
1994
). The large outward current at membrane potentials more
positive than
60 mV was abolished, and inward currents, probably
voltage-gated Na+ or Ca2+
currents, were revealed (Fig. 2E). However,
Erev for the linear current only
shifted to
1.8 ± 2.7 mV (n = 6; Fig.
2F).
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Ca2+-dependent nonselective cationic currents
with linear I-V relationships have been demonstrated in
DRGs, and these are activated by Ca2+ release
from internal stores (Currie and Scott 1992). Therefore we tested the effects of OaV in the presence of
thapsigargin, a membrane-permeant Ca2+-ATPase
inhibitor that prevents the refilling of intracellular Ca2+ stores (Verkhratsky and Shmigol
1996
). OaV was applied twice to DRGs (~7-min
interval between applications), and the ratio of the second to the
first response was calculated (average response = 1/To
TIdt,
where T is the duration of the response). In controls the ratio of the second to the first response was 2.69 ± 0.46 (n = 9; Fig.
3A). These data were compared
with the ratio obtained from successive applications of OaV
when 1 µM thapsigargin was applied extracellularly (Fig.
3B). In the presence of thapsigargin the inward current in
response to the first OaV application was similar to
controls, presumably because the intracellular stores still contained
some Ca2+. However, the second was very small,
and as a result the ratio of the second to the first response fell to
0.17 ± 0.09 (n = 5; Fig. 3B).
Thapsigargin did not wash out as demonstrated by the lack of response
to a third application of OaV (Fig. 3B). The inward current induced by OaV was also blocked by 200 nM
k252a, a tyrosine and serine-threonine kinase inhibitor at this
concentration (ratio of 2nd to the 1st response = 0.44 ± 0.16, n = 4; Fig. 3C). The response to
OaV was restored after wash out of k252a, which is
consistent with the known reversibility of some actions of this drug
(Knight et al. 1997
). Comparison of the ratio responses in controls, thapsigargin- and k252a-treated cells (Fig. 3D)
revealed a highly significant effect of treatment (1-way ANOVA,
P < 0.001) and significant differences between
controls and both other treatment groups (Dunnett's test,
P = 0.025). Although the concentration of thapsigargin
used may block high-voltage-activated and, to a lesser extent,
low-voltage-activated Ca2+ currents in DRGs
(Rossier et al. 1993
; Shmigol et al.
1995
), this is unlikely to be its route of action because only
the response to a second application of OaV was reduced.
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DISCUSSION |
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This study is the first to report an action of an animal venom on
isolated sensory neurons. We believe that a high proportion of the
cells from which we recorded were probably nociceptors. To prepare
cultured DRG neurons from adult rats, we used a procedure very similar
to that described by Gold et al. (1996). These authors reported that about 60% of neurons responded to capsaicin, and we
report a similar figure. A further 16% of all DRGs express a
structural homologue of VR1 that has a high-threshold for noxious heat
but is capsaicin insensitive (Caterina et al. 1999
).
Thus we can reasonably expect that around 80% of our recordings were from cell bodies of nociceptive neurons, and the figure may be higher
because we tried to select smaller diameter cells, which are those
associated with nociception in vivo (Harper and Lawson 1995
).
We chose to use cultured DRGs as model nociceptors to minimize the
amount of venom required for each experiment. Lack of nerve growth
factor in culture medium is known to lead to the progressive loss of
proton and capsaicin sensitivities over several days (Bevan 1996). Therefore we used the cultured DRGs within 6-24 h of
plating, and, accordingly, the proton and capsaicin sensitivities
remained robust.
A very short application of OaV to putative nociceptors
produced a long-lasting inward current. The possibility that this current reflects cell damage (due, for example, to protease activity in
the venom) can be excluded for two reasons. First, when the current
eventually ceased, the holding current was usually similar to that
prior to application of OaV, and, second, when
OaV was applied twice with a short interval between
applications, the response to the second test was bigger than that to
the first. It is also unlikely that the observed responses arise from
venom-induced metabolic inhibition because they are completely
different from those reported in DRGs under such circumstances
(Duchen 1990).
If the inward current occurred in vivo, it would be expected to lead to
firing and the perception of pain. This correlates well with reports of
immediate and intense pain after platypus envenomation of humans
(Fenner et al. 1992). The pH sensitivity of the response
is notable given that an interaction between various pain-producing
molecules and pH has previously been reported (Kress et al.
1997
; Petersen and Lamotte 1993
; Tominaga
et al. 1998
) and that inflammatory exudates are acidic in
nature. We have measured the pH of platypus venom and found it to be
6.3. Although this suggests that the venom component, or components,
activating nociceptors in vitro, could probably do so in vivo,
interstitial fluids may buffer the venom at a higher pH.
Our data indicate that OaV principally activates a
nonselective cationic current that is probably dependent on
Ca2+ release from intracellular stores. It is
somewhat surprising that such a current could be activated with 11 mM
EGTA in the recording pipette. However, when using a pipette solution
of similar composition to that used here, and additionally containing
IP3, Li and Zhao (1998) reported
induction of a long-lasting current in DRGs, characterized by transient
events and a very long latency to onset (2-4 min). Clearly,
Ca2+ released from intracellular stores was not
completely buffered by the high concentration of EGTA. The long
latencies to onset (about 23 s) of the OaV- or
IP3-induced responses do not appear to be the
result of Ca2+ buffering either because responses
to the metabotropic glutamate receptor agonist,
1-amino-1,3-cyclopentanedicarboxylic acid [(1S,3R)-ACPD], develop
with latencies of 2-4 min when only 0.1 or 1 mM EGTA is used in the
pipette solution (Crawford et al. 1997
). Responses to
ryanodine and caffeine also develop with very long latencies (Ayar and Scott 1999
).
Our conclusion that the OaV-induced response is
Ca2+ dependent arises largely from its block by
200 nM thapsigargin. This conclusion is consistent with the fact that
in DRG neurons transient events imposed on transmembrane currents are
only associated with agonists known to release
Ca2+ from intracellular stores, such as
1S,3R-ACPD, ryanodine, caffeine, and cGMP, and not with capsaicin or
low pH. It is also important to note that
Ca2+-dependent nonspecific cationic currents have
been described in a wide variety of tissues and they have linear
I-V relationships between 100 and
60 mV
(Partridge et al. 1994
). Thapsigargin at 200 nM
concentration is not detrimental to sensory neurons, although it does
depress high-voltage-activated Ca2+ currents by
about 40% (Shmigol et al. 1995
). The block of the OaV-induced current could not be attributed to an affect on
the latter since the first response to OaV in the presence
of thapsigargin was similar to that in its absence.
The results presented suggest that the venom-induced release of
Ca2+ from intracellular stores involves
activation of tyrosine or serine-threonine kinases. The tyrosine
kinase, TrkA, binds nerve growth factor (NGF) with high affinity, and
NGF evokes increases in cytosolic Ca2+ in both
TrkA-expressing C6-2B glioma cells (De Bernardi et al. 1996) and in 3T3 cells (Jiang et al. 1999
).
Interestingly, we have found that OaV contains an NGF
(de Plater et al. 1998a
) and NGF forms a substantial
component of snake venoms (Kostiza and Meier 1996
). The
response to subcutaneous injection of recombinant NGF in humans has not
been measured any earlier than 3 h postinjection, but at this time
it caused pressure allodynia and lowered the heat-pain threshold in a
significant percentage of subjects (Dyck et al. 1997
).
Eventually, most subjects experienced allodynia and hyperalgesia that
lasted 21-27 days (Dyck et al. 1997
; Petty et
al. 1994
). The nature and time course of these symptoms bear a
striking similarity to those reported after platypus envenomation (Fenner et al. 1992
).
However, the acute activation of an inward current in DRGs by
OaV is not consistent with the known actions of NGF and may therefore depend on activation of a serine kinase that induces intracellular Ca2+ release, for example, protein
kinase C (PKC). PKC-epsilon has been implicated in the sensitization of
the noxious heat response by bradykinin (Cesare et al.
1999) and PKC-gamma in neuropathic pain (Malmberg et al.
1997
). Further studies are required to elucidate the signal
transduction mechanisms involved in the response we have reported.
Platypus venom contains many constituents (de Plater et al.
1995), and we have commenced studies to identify the component or components responsible for activating an inward current in DRG
neurons. We can exclude involvement of the C-type natriuretic peptide
(OaV-CNP) present in the venom (de Plater et al.
1995
) because neither CNP purified from whole venom nor
synthetic CNP induced a current in DRG neurons (data not shown), even
though the synthetic form is capable of forming channels in artificial membranes (Kourie 1999
). The venom has some protease
activity that is associated with high molecular weight venom components (de Plater et al. 1995
). In preliminary studies we have
tested the activity of gel filtration/HPLC fractions on DRG neurons and have found that the fractions capable of inducing the inward current are of much lower molecular weight. Furthermore, if the
OaV-induced current resulted from irreversible proteolytic
cell damage, then recovery of the current to baseline, as observed in
56 of 69 neurons, or repeated responses from the same cell would be
unlikely. The venom is known to contain four defensin-like peptides
(Torres et al. 1999
, 2000
), and these
have some structural similarity with the sodium neurotoxin peptide, ShI
(Torres et al. 1999
). Defensins are also capable of
forming channels in membranes (reviewed by Kourie and Shorthouse
2000
). However, Torres et al. (1999)
reported
that defensin-like peptide 1 had no effect on dorsal root ganglion
sodium currents, and none of the platypus defensin-like peptides have
antimicrobial, myotoxic, or cell growth-promoting activities
(Torres et al. 2000
). Another possible explanation for
the responses we have observed in DRG neurons is that the venom
contains free glutamate. While we have not established whether this is
the case, the HPLC elution profile of active fractions suggests that
glutamate is not responsible for the activity.
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ACKNOWLEDGMENTS |
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Our special thanks go to the Australian Platypus Conservancy for supply of venom.
This work was supported by the Clive and Vera Ramachiotti Foundation, the Australian Research Council, and the John Curtin School of Medical Research.
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FOOTNOTES |
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Address for reprint requests: R. L. Martin (E-mail: Rosemary.Martin{at}anu.edu.au).
Received 7 March 2000; accepted in final form 20 November 2000.
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REFERENCES |
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