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Lophotoxin: Selective blockade of nicotinic transmission in autonomic ganglia by a coral neurotoxin
NeutoscienceVol. 20, No. 3, pp. 875-884, 1987 Printed in Great Britain
03~~522/87 %3.00+ 0.00 Pergamon Journals Ltd 0 1987 IBRO
LOP~OTOXIN: SELECTIVE NICOTINIC TRANSMISSION GANGLIA BY A CORAL
BLOCKADE OF IN AUTONOMIC NEUROTOXIN
EVA M. SORENSON,* PAUL CULVER? and VINCENT A. CHIAPPINELLI*$ *Department of Pha~acology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A.; TDivision of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A. Abstract-Lophotoxin is a diterpene lactone isolated from gorgonian corals. The toxin has previously been shown to bind with high affinity to an acetylcholine recognition site located on skeletal muscle nicotinic receptors, producing an essentially irreversible blockade of neuromuscular transmission. Lophotoxin has also been shown to block nicotinic transmission in autonomic ganglia of the frog and in ileal strips of guinea pig and rabbit. The effects of lophotoxin have now been examined on neuronal nicotinic receptors in autonomic ganglia of the chick and rat. Low concentrations of lophotoxin (1 PM) produce a blockade of neuronal nicotinic transmission which is partially reversed by 3-5 h of washing out the toxin. The blockade produced by higher concentrations of lophotoxin (up to 32 PM) is not reversed during a similar washout period. Prior exposure to d-tubocurarine, a competitive nicotinic antagonist, can partially protect ganglia against exposure to lophotoxin. In contrast the local anesthetic QX-314, a noncompetitive nicotinic antagonist, does not protect ganglia against lophotoxin exposure. Lophotoxin binds to a site in ganglia identified by [lzsI]K-bungarotoxin which appears to be on the neuronal nicotinic receptor. Intracellular recordings reveal that lophotoxin has no effect on either muscarinic responses or on responses to y-aminobutyrate in autonomic ganglia. Passive and active membrane properties of the neurons are unaffected by lophotoxin except for the blockade of nicotinic responses. It is concluded that lophotoxin is a selective, high-affinity antagonist at the neuronal nicotinic receptor. The long-term nature of the blockade with lophotoxin suggests that the toxin will be of considerable value as a probe for characterizing the ganglionic nicotinic receptor.
Nicotinic acetylcholine receptors are present in vertebrate nervous tissue in both autonomic ganglia and the central nervous system. These neuronal nicotinic receptors share some characteristics with the nicotinic receptors found in skeletal muscle and in the muscle-
derived electric organs of certain electric fish. While nicotine is a potent antagonist of acetylcholine action at nicotinic receptors in both ganglia and muscle, other drugs can be used to differentiate these two receptor subtypes. For example, trimethaphan and hexamethonium are potent inhibitors of neuronal nicotinic receptors, but are active at muscle receptors o&y at much higher concentrations.38 The most important ligands for characterizing muscle and muscle-derived nicotinic receptors have been the cc-neurotoxins isolated from the venom of various elapid and hydrophid snakes.2’ These basic poly~ptides (Mr = 65~-gOO0) bind with extremely high affinity (& = 10-9-10-‘2 M) and specificity to the acetylcholine recognition sites on muscle receptors.4.20a-Bungarotoxin, purified from the venom of Bungarus multicinctus, is one of the most potent of these E-neurotoxins in blocking muscle receptors. Yet most a-neurotoxins, including tr-bungarotoxin, demonstrate little or no physiological activity at a number of neuronal nicotinic receptors2.3,6 (but see $Author to whom correspondence should be addressed. Abbreviation: GABA, y-aminobutyrate.
also Refs 18 and 28). A related family of snake toxins, termed the K-neurotoxins, block neuronal nicotinic receptors at doses well below those required for blockade of muscle receptors.5a**The characteristics of the blockade support the conclusion that Ic-neurotoxins bind with high affinity to acetylcholine recognition sites on neuronal nicotinic receptors.s~7~‘4 Radiolabeled derivatives of Fc-neurotoxins, such as [1251]K-bungarotoxin, can thus be used as probes for the neuronal nicotinic receptors5 Few other ligands are available which can be similarly used. Recently the structure of lophotoxin, a diterpene lactone isolated from gorgonian corals, has been described.” At doses of 2-32 PM, this toxin produces an essentially irreversible blockade of nicotinic neuromuscular transmission.‘.‘3,23 Intracellular electrophysiological studies demonstrate that lophotoxin blocks miniature endplate potentials, endplate potentials, and the direct actions of acetylcholine in skeletal muscle cells while having very little or no effect on prejunctional nerve terminals.‘.23 Binding studies reveal that lophotoxin blocks transmission by binding to the acetylcholine recognition sites on muscle nicotinic receptors.‘* At concentrations of 1632 p M, lophotoxin also blocks nicotinic transmission in frog sympathetic ganglia and rabbit and guinea pig ileal strips in an apparently irreversible manner.24 The present study was undertaken to further char875
EVA M. SORENSON et al.
876
Control
Response
Ciliary Ganglion
,_,J
n
\ Y&*
1% DMSO 60 min
1==== i__
t/ 32 FM Lophotoxin
32 FM Lophotoxin
6 min
15 min I
r--
I---------
32 FM Lophotoxin
Wash
30 min
3.8 hr
L _-h
---
--L__-
Fig. I. Effect of lophotoxin on transmission in the chick ciliary ganglion. Electrical stimulation of the preganglionic nerve (break in record) results in three separate compound action potentials, recorded extracellularly from the postsynaptic ciliary nerve. The response with the shortest latency (arrow) represents ciliary neurons activated by electrical coupling potentials. The largest response is due to ciliary neurons activated chemically through nicotinic receptors. The small response with the longest latency represents choroid neurons activated by nicotinic receptors. Exposure to I% (v/v) dimethyl sulfoxide (DMSO) had no detectable effect on the cells. The flow was stopped during this 60 min incubation with 1% dimethyl sulfoxide, as it was during exposures to toxins. Exposure to 32 p M lophotoxin completely blocked nicotinic transmission in both ciliary and choroid cells in 30 min. This effect was not reversible over the time period examined, which ranged up to 5 h after washing out the toxin. Note that lophotoxin had no effect at any time on electrically mediated synaptic transmission. Calibration bars: 1 mV and 2 ms.
acterize
the
effects
ganglia,
since
the
of
lophotoxin
toxin
exhibits
which would be desirable nicotinic
receptor.
ently selective tors,
and
methods sponses in
Intracellular on
neurons
tection
been
action
within
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petitive
and
Finally,
the ability
identified
of
extracellular
ciliary
and
nicotinic
by [‘251]~-bungarotoxin
re-
(GABA) sympathetic
ganglion.
have been performed of lophotoxin
the
lophotoxin’s
muscarinic
cervical
non-competitive
to
recording
to y-aminobutyrate
chick
recep-
binding
transmission,
and the rat superior experiments
include an apparon nicotinic
used to determine
nicotinic
and responses
ganglia
properties
in a probe for the neuronal
and potent
have
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autonomic
These properties
a prolonged
receptors.
in several
Pro-
with comantagonists.
to bind
to sites
has been examined.
EXPERIMENTAL PROCEDURES Methods for intracellular”” and extracellularS recording from isolated, intact autonomic ganglia have previously been described. Briefly, for extracellular recording, suction electrodes were used to stimulate preganglionic nerve trunks at 1Hz and record the resulting compound action
potentials in postganglionic nerve trunks. Responses were differentially amplified (Tektronix 5A22N) and displayed on an oscilloscope. Intracellular recordings were made by standard bridge-balance methods using a WPI-M707 electrometer. Glass microelectrodes pulled on an Industrial Science Associates (Ridgewood, New York) electrode puller were filled with 3 M KC1 and had resistances of 60-120 MD. Ciliary ganglia and lumbar sympathetic chain ganglia were dissected from 18-20 days of incubation White Leghorn chick embryos (SPAFAS, Peoria, IL). Superior cervical ganglia were removed from 150-200 g SpragueDawley rats. All ganglia were desheathed prior to electrophysiological recordings, which were performed either at 22°C (extracellular recording) or at 35°C (intracellular recording). The physiological saline used was of the following composition: NaCl (150mM); KC1 (3 mM); CaCl, (5 mM); MgCI, (2 mM); glucose (17 mM); HEPES (IO mM); pH 7.4, bubbled with 100% 0,. The ganglia were perfused with physiological saline at 24 ml/min. The flow was stopped during exposures to lophotoxin and h--bungarotoxin in order to conserve the toxins. Control experiments indicated that 60-90min periods of stopped flow in toxin-free physiological saline had no effect on ganglia neurons (see Fig. 1). Pressure injections of carbachol and GABA were performed with a small pressure pipette placed 3 mm “upstream” from the ganglion.‘4 IO ~1
Lophotoxin blocks neuronal nicotinic transmission of drug (lOa M) dissolved in physiological saline was injected while maintaining the 2-4 mi/min flow rate in the recording chamber, which had a total capacity of 1.7mi. Binding experiments were performed at 22°C on freshly dissected whole chick ciiiary ganglia as previously described.5 Ganglia were placed into plastic microwells with fine forceps. The incubation buffer (final volume = 30 ~1) was identical to the physiological saline described above, with the addition of 1mg/mi bovine serum albumin. Preincubations with various concentrations of drugs were for 1 h, followed by a 2 h incubation with added [Y]Kbungarotoxin (final concentration = 20 nM). At the end of this incubation period, ganglia were washed with several rinses of 4°C incubation buffer for a total of 10min. Individual ganglia were then placed in vials and bound radioactivity was determined in a y-counter. Control experiments demonstrated that 1% (v/v) dimethyl sulfoxide had no effect on [‘251]K-bungarotoxin binding. Materials Crystalline iophotoxin was prepared according to the method of Fenicai et al.” Due to the hydrophobic nature of the toxin, stock solutions of IOmM lophotoxin were prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide to which ganglia were exposed never exceeded 1% (v/v). Control experiments (see Fig. 1) indicated that 1% dimethyl sulfoxide had no effect on the physiological properties of ganglia neurons. In a recent study, similar concentrations of dimethyl suifoxide did not interfere with transmission in bullfrog sympathetic ganglia, although higher concentrations (3-10%) produced a facilitatory effect on transmission.)’ K-Bungarotoxin and a-bungarotoxin were purified from the crude venom of Bungarus multicinctus(Miami Serpentarium, Salt Lake City, UT, Lot No. BM&lOSTLZ) as reported.’ QX-314 and QX-222 were the generous gifts of Astra Lakemedel AB, Sodertalje, Sweden. Other drugs and chemicals were obtained from Sigma Chemicals, St. Louis, MO. RESULTS
Blockade of nicotinic transmission by lophotoxin In isolated, intact ciliary ganglia, electrical stimulation of the preganglionic oculomotor nerve elicited up to three distinct compound action potentials, recorded extracelluiariy from the postsynaptic ciliary nerves (Fig. 1). The shortest latency response was due to ciliary neurons which were activated by electrical coupling potentials. 29 The largest response represented ciliary neurons activated chemically via nicotinic acetylcholine receptors. In some ganglia, a third peak with a longer latency was observed and was due to choroid neurons activated by nicotinic receptors.30 At a dose of 32 PM, lophotoxin completely blocked nicotinic transmission in 30min in both ciliary and choroid neurons (Fig. I). Following exposure to 32 ,UM iophotoxin, no recovery was seen in nicotinic transmission in the ganglia for up to 5 h after washing out the toxin (Fig. 1). The compound action potential representing ciliary neurons activated by electrical synaptic transmission was unaffected by iophotoxin. Lower doses of lophotoxin required considerably longer periods of exposure to obtain a complete blockade. The lowest dose to achieve a complete block of nicotinic transmission in 90 min was 1 FM lophotoxin (Fig. 2). Following exposure to low doses
877
of lophotoxin, a partial recovery of nicotinic transmission was seen (Fig. 2). This recovery required at least 3 h of washing out of lophotoxin. Lophotoxin also blocked nicotinic transmission in the rat superior cervical ganglion, although higher doses of lophotoxin were required for a complete blockade in the rat. For example, after 30min at 3 PM lophotoxin, nicotinic transmission in chick ganglia was completely blocked, while rat ganglia were totally unaffected. A dose of 32 PM iophotoxin did completely block nicotinic transmission in rat ganglia after an exposure of 1 h. Protection experiments
Previous studies have demonstrated that the reversible competitive antagonist d-tubocurarine can be used to protect autonomic ganglia against the long-term blockade produced by ~-bungarotoxin.’ A similar experiment was carried out with lophotoxin. Pre-exposure of ciliary ganglia to 300 p M d-tubocurarine partially protected against the subsequent addition of 3 PM lophotoxin (Fig. 3). Virtually identical results were obtained in two different ganglia using 3OOpM d-tubocura~ne. This dose of d-tubocurarine was approximately &fold higher than the concentration required for a complete blockade of nicotinic transmission in the ganglia. A ten-fold lower dose of d-tubocurarine (30pM) did not protect against subsequent exposure to 3 PM lophotoxin in another ganglion. Doses of d-tubocurarine higher than 300,uM were not tried due to the possible nonselective effects of the drug at higher concentrations. Local anesthetics have been shown to block the actions of acetylcholine at nicotinic receptors in a noncom~titive fashion. lo These compounds appear to block the ion channel associated with the nicotinic receptor. 22,34Two lidocaine derivatives were selected to determine whether local anesthetics could protect against the long-term blockade caused by lophotoxin. The use of the quaternary lidocaine derivatives, QX3 14 (quaternary N-ethyl) and QX-222 (quaternary ~-methyl), kept nonspecific absorption of drug into ceil membranes at a minimum, providing rapid washout times. QX-314 produced a complete blockade of nicotinic transmission at 100 PM in chick ciliary ganglia and was approximately three-fold more potent than QX-222. Complete recovery from QX314 exposure required a washout period of between 10 and 40 min. As with the d-tubocurarine protection experiments, the QX-314 concentration was varied between the minimum required for a blockade (100 PM) and an 8-fold higher dose in a series of protection experiments in four separate ganglia. Axonal conduction was not impaired by these concentrations of QX-314, since electrically mediated ciliary neuron responses remained intact (Fig, 4B). Even at the highest dose tested (800 PM) QX-314 did not protect ganglia against the subsequent addition of 3 PM iophotoxin, as after a 3 h washout of both
EVA M. SORENSON et al.
878
Control Ciliary
Response
1 PM Lophotoxin
Ganglion
27 min
tn
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1 FM Lophotoxin
60 min
90 min .-,,
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-
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Wash
47 min
5.3 hr
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Fig. 2. Lophotoxin blockade is partially reversible. Preganglionic stimulation elicits electrically mediated ciliary neuron response (arrows; partially obscured by stimulation artifacts), chemically mediated ciliary neuron response (largest response) and chemically mediated choroid neuron response. Both nicotinic responses are completely blocked after exposure to I PM lophotoxin for 90 min, while the electrical response is unaffected. Washout of the toxin began after 90 min exposure. Recovery of the chemically mediated responses began to appear at 3 h of washout, and by 5.3 h of washout, both ciliary and choroid responses were recognizable. Calibration bars: 0.5 mV and 2 ms.
toxins no nicotinic transmission could be detected (Fig. 4A). Similarly, pre-exposure of ganglia to QX314 at either 100pM or 800pM was unable to protect against the subsequent addition of 100nM K-bungarotoxin (Fig. 4B).
Intracellular
recording
Intracellular recording was used to further examine the nature of the blockade seen with lophotoxin. In these experiments, chick lumbar sympathetic chain ganglia were continuously perfused with physiological saline containing 1.5 PM atropine. This dose of atropine was sufficient to completely block muscarinic responses in the ganglia, while leaving nicotinic responses intact.’ Under control conditions in the presence of atropine, carbachol produced a robust depolarization associated with a decrease in input resistance (Fig. 5A). A similar response was seen following exposure to GABA (Fig. 5A). The
GABA response was mediated by a bicuculline- and picrotoxin-sensitive GABA, receptor.j2 Following exposure to lophotoxin, the nicotinic response to carbachol was almost completely blocked, while the GABA response was unchanged (Fig. 5B). After lophotoxin exposure, neurons exhibited normal resting membrane potentials and displayed overshooting action potentials in response to direct stimulation through the intracellular electrode (Fig. 5B). Similar results were obtained from a total of 11 neurons after lophotoxin exposure in three different sympathetic ganglia preparations. To determine whether muscarinic responses were affected by exposure to lophotoxin, atropine was deleted from the physiological saline. Under these conditions, the muscarinic response to carbachol (a depolarization with an increase in input resistance15) persisted after lophotoxin exposure (Fig. SC). Similar results were seen in three different cells from two different preparations.
Lophotoxin blocks neuronal nicotinic transmission
Control Response
300
Ciliary Ganglion
i
879
PM d-Tubocurarine + 3 pM Lophotoxin
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16 min
2.4 hr
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Wash 2 hr
Fig. 3. d-Tubocurarine protects against lophotoxin blockade. When exposed to 300 PM d-tubocurarine for 50 min, chemical transmission in ciliary neurons is completely blocked in 5 mitt, and complete recovery requires 20 min of washout (not shown). The protection experiment consists of a 10 min exposure to d-tubocurarine, followed by a 40 min exposure to d-tubocurarine and 3 PM lophotoxin. Washout is for Smin with 3OOgM d-tubocurarine, followed by continued washout with normal physiological saline. Partial recovery from exposure to both toxins is seen after 16 min, and the recovery is slightly increased in size by 2.4 h of washout. Finally, 3 FM lophotoxin is applied to the ganglion for 25 min. A complete block of the remaining response occurs in II min, and no recovery is seen after 2 h of washing out the toxin. Calibration bars: 0.2 mV and 2 ms. Binding experiments
was blocked by IO PM lophotoxin, while 30~1cM lophotoxin blocked 99% of this specific binding [‘*‘I]~-Bungarotoxin binds to two different phar(Fig. 6). Similar results were obtained in three differmacologically nicotinic sites in chick ciliary ganglia.5 Most (84%) of the specific binding of [1251]rc- ent binding experiments. Thus, lophotoxin and ?c-bungarotoxin bind to the same nicotinic sites in bungarotoxin is to a site which is also detected ciliary ganglia, a portion of which are associated with by the physiologically inactive a-bungarotoxin the neuronal nicotinic receptor. (Fig. 6). Considerable electrophysiologicals~6~‘4~2s and anatomical26 evidence indicates that this neurotoxin DISCUSSION binding site is not associated with ganglionic nicotinic transmission, The remaining 16% of specific The present results support the conclusion that [‘251]?c-bungarotoxin binding is not blocked by 1 PM lophotoxin blocks nicotinic transmission in autox-bungarotoxin (Fig. 6) but is blocked by the nico- nomic ganglia by binding to acetylcholine recognition tinic receptor affinity agent bromoacetylcholine.5 This sites on neuronal nicotinic receptors. An identical binding site appears to be located on the physio- mechanism has previously been proposed to account logically detected neuronal nicotinic receptor.5.25~“7 To for lophotoxin’s action at the neuromuscular juncdetermine whether lophotoxin binds to either of these tion.‘,‘2,23The selectivity of lophotoxin for nicotinic [‘251]~-bungarotoxin sites, ganglia were pre-exposed receptors was demonstrated by its inability to block to two different concentrations of lophotoxin. Most muscarinic or GABAergic receptors in autonomic of the ]‘251]rc-bungarotoxin specific binding (85%) ganglia.
880
EVA M. SORENSON et al
A Control Ciliary
800
pM QX 314 + 3 IJM Lophotoxin
Response Ganglion
800
B Control Response Ciliary Ganglion
+
pM QX 314
100 nM K-Bungarotoxin
Wash
Wash
2.2 hr
3.2 hr
Wash 2.0 hr
L
Wash 3.3 hr
Fig. 4. Lidocaine derivative QX-314 does not protect against lophotoxin blockade. Protocol for protection experiment is identical to that described for d-tubocurarine protection experiment (legend to Fig. 3) except that 800nM QX-314 is substituted for d-tubocurarine. Complete recovery from exposure to 800 PM QX-314 alone required 40 min of washout (not shown). (A) 800 PM QX-314 does not protect against exposure to 3 PM lophotoxin, since no recovery is seen after 2.2 h and 3.2 h of washing out the toxin. (B) 800pM QX-314 does not protect against exposure to 100 nM h--bungarotoxin. Note that the electrically mediated ciliary neuron response (arrow) is unaffected by either QX-314 or h--bungarotoxin. The recovery from K-bungarotoxin exposure begins at 3.3 h of washout, which is similar to recovery times following exposure to x-bungarotoxin alone.s Calibration bars: (A) 0.2 mV and 2 ms; (B) 0.5 mV and 2 ms. Extracellular electrophysiology A single study has previously characterized the effects of lophotoxin in ganglionic preparations.24 At a high dose (1632 p M), lophotoxin blocked nicotinic responses recorded extracellularly in frog sympathetic ganglia and in ileal strips from rabbit and guinea pig. In the frog ganglia, no recovery was seen after 16 h of washing out the toxin. The effects of lower doses of lophotoxin were not examined. No protection experiments were undertaken in this study, nor were any intracellular recordings made. In the present study, extracellular recordings from chick and rat autonomic ganglia revealed an apparently irreversible blockade of nicotinic transmission at high concentrations (32 p M) of lophotoxin. At lower doses (1 PM) of the toxin, however, the blockade was at least partially reversible after 3-5 h of washing out the toxin. Thus, it is not clear whether any effects of lophotoxin in ganglia are in fact “irreversible”. Given sufficient time, the ganglia might also have recovered from the higher doses of the toxin. Lophotoxin produces an apparently irreversible blockade of nicotinic transmission in a variety of in neuromuscular preparations.‘~‘2~“~23~24 Results BC3H-1 cells (a muscle cell line) are consistent with lophotoxin binding in a two-stage process to the muscle nicotinic receptor. ‘* The initial binding is of relatively low affinity and is reversible. However, with continued exposure to the toxin, the binding proceeds to an apparently irreversible stage. Such a process
may explain why recovery ganglia only at low doses transition to irreversible occurred by the end of the
was seen in autonomic of lophotoxin, where a binding may not have exposure period.
Protection experiments The long-term blockade of nicotinic transmission produced in chick ganglia by 3 FM lophotoxin could be partially prevented by prior exposure of ganglia to the competitive nicotinic antagonist d-tubocurarine. d-Tubocurarine binds selectively to acetylcholine recognition sites on nicotinic receptors.” At the highest dose of d-tubocurarine used (300pM), approximately 40% of the nicotinic response was protected. Lower doses (30 PM) were not effective in protecting against lophotoxin. The reasons that relatively high doses of d-tubocurarine were required and only a partial protection was seen are not known, although the slow off-rate of lophotoxin likely contributed to this difficulty in protecting against the toxin. In addition, lophotoxin is strongly lipophilic, and during the washout period, toxin molecules dissolved in ganglion cell membranes may diffuse and become associated with nicotinic receptors at a time when d-tubocurarine is no longer present in solution. Evidence for such a phenomenon has been reported in intact BC3H-1 cells.‘* The protection against lophotoxin afforded by d-tubocurarine is due to the two toxins interacting at identical sites, presumably acetylcholine recognition sites on neuronal nicotinic receptors, Local anes-
Lophotoxin
blocks
A. Control Responses
neuronal
nicotinic
881
transmission
in 1.5 FM Atropine
Carb
GABA
B. Responses After Lophotoxin Carb
Exposure
in 1.5 PM Atropine
GABA
50
mv 0.5
0.7
“AI
__
min
-
1
C. Muscarinic
Response
Unaffected
Carb
GABA
+
$
by Lophotoxin
0.5
min
Fig. 5. Intracellular records from chick lumbar sympathetic ganglia. (A) In the presence of 1.5pM atropine, pressure injection of carbachol (Carb; 10~1 of 10e2 M) produces a depolarization in a sympathetic cell. This depolarization is associated with a decrease in input resistance, measured as the voltage response to constant pulses of hyperpolarizing current. y-Aminobutyrate (GABA; 10~1 of 10-2M) also produces a depolarization with a decrease in input resistance. (B) The ganglia are subsequently exposed to 32 PM lophotoxin for 20 min. Approximately 30 min after lophotoxin exposure, the response to carbachol (10 yl of 10e2 M) in another cell is almost completely blocked. Response to GABA (IO ~1 of 10m2 M) is similar to that seen before lophotoxin exposure. A depolarizing current pulse delivered after recovery from GABA (asterisk) is also shown on a greatly expanded time scale (lower record). The cell exhibits normal-appearing, overshooting action potentials in response to the depolarizing current pulse. (C) To determine whether lophotoxin has any effect on muscarinic responses in sympathetic ganglia, atropine was deleted from the physiological saline in another preparation. Following exposure to lophotoxin (32 PM for 20 min), the response to carbachol(10 ~1 of 10e2 M) consists of a depolarization associated with an increase in input resistance. This response is muscarinic in nature.15 y-Aminobutyrate (10 ~1 of 10m2 M) produces a depolarization with a decrease in input resistance similar to that seen before lophotoxin exposure. Resting membrane potential for the neurons shown is between -40 mV and - 50 mV.
thetics,
which
are
noncompetitive
nicotinic
antag-
do not bind to these acetylcholine recognition sites,‘0,22,34do not protect chick ganglia from exposure to 3 p M lophotoxin. Similar results have previously been obtained for the muscle nicotinic receptor of BC3H-1 cells.” In this preparation, competitive agonists and antagonists such as carbachol, pancuronium and d-tubocurarine protect against the action of lophotoxin, while the local anesthetic dibucaine is unable to prevent the lophotoxin blockade. onists
that
Intracellular recording Intracellular records from chick autonomic ganglion cells reveal that lophotoxin has a selective effect on postsynaptic neurons. Nicotinic responses to carbachol are almost completely blocked by the toxin at concentrations where muscarinic and GABAergic responses are unaffected. Langdon and Jacobs” report that lophotoxin is also ineffective in blocking muscarinic receptors located in smooth muscle. Thus, the blockade of nicotinic receptors produced by lophotoxin is selective, and is not accompanied by
882
EVA M. SORENSON Edal
q
Total Binding
Lophotoxin
1 FM KBgT 1 pM ABgT
*
Fig. 6, Inhibition of [‘*jI]K-bungarotoxin binding in ciliary ganglia by lophotoxin. Intact ciliary ganglia were pretreated for 1 h with lophotoxin (IOpM or 30,~M), x-bungarotoxin (KBgT: 1 FM), ~-bungarotoxin (ABgT; 1PM), or no toxin (Total Binding), followed by a 2 h incu~tion with 20 nM [1251J~-bungarotoxin. Bars represent mean f S.E.M. (N = 5 in each group). Percentages reported are remaining amount of specific binding (defined as Total Binding minus binding in the presence of 1 pM h--bungarotoxin). *P -C0.025 between binding in the presence of 1PM a-bungarotoxin and binding in the presence of 1 PM K-bungarotoxin.
K-neurotoxins and bromoacetylcholine,’ but is not detected by the inactive a-bungarotoxin. This site appears to be located on the ganglionic nicotinic receptor.s.25+27Lophotoxin inhibits the binding of [‘251]~-bungarotoxin to this selective K-neurotoxin site. inhibition of K-neurotoxin binding Two independent experiments thus confirm that lophotoxin is a competitive antagonist at neuronal Snake venom rc-neurotoxins are potent competitive antagonists at ganghonic nicotinic receptors.” This nicotinic receptors. First, the competitive antagonist class of peptide toxins includes K--bungarotoxin,‘.” d-tubocurarine protects against lophotoxin action. all purified from Bungurus Second, lophotoxin inhibits the binding of [‘251]~Toxin F?’ and Bgt 3. I, 35~36 ~~it~ri~~t~~ venom, and K-flavitoxin’ from the bungarotoxin to a nicotinic site which is located on venom of ~~ngarus Jtaviceps. Preliminary evidence the neuronal nicotinic receptor. Lophotoxin should indicates that the amino acid sequence of Toxin F17 therefore be a useful probe in characterizing neuronal may be identical to the complete amino acid sequence nicotinic receptors. The very slowly reversible or reported for K-bungarotoxin.iY Radiolabeled derivairreversible nature of iophotoxin binding to neuronal tives of ~-bungarotoxin,* Toxin F2’ and rc-ffavitoxin’ nicotinic receptors indicates that radiolabeled lophobind to two nicotinic sites in autonomic ganglia. One toxin may be an important Iigand in the identification of these sites, which is also detected by radiolabeled and purification of this receptor. The high lipid a-bungarotoxin, is not associated with neuronai nico- solubility of lophotoxin may pose problems with high tinic transmission.6 The second site is identified by nonspecific binding. Fortunately, several congeners physiologicaily active compounds such as the of lophotoxin are also active at nicotinic receptors,” any other observable effects on the active or passive membrane properties of the cells. In particular, the cells continue to maintain a normal resting membrane potential and continue to fire action potentials in response to injected depolarizing current.
Lophotoxin suggesting found
for
blocks
neuronal
that a less lipophilic derivative may be binding
experiments.
Acknowledgements-This research was supported by the National Institutes of Health, Research Grant NS 17574 to
nicotinic
transmission
883
V.A.C. We wish to thank Kathleen Wolf for expert technical assistance and preparation of the figures, and Maggie Klevorn for preparation of the manuscript. We are grateful to Dr Bengt Akerman of Astra Lakemedel AB for providing investigational samples of QX-314 and QX-222.
REFERENCES 1. Atchison
W. D., Narahashi T. and Vogel S. M. (1984) Endplate blocking actions of lophotoxin. Br. J. Pharmac. 82, 667-672. 2. Brown D. A. and Fumagalli L. (1977) Dissociation of a-bungarotoxin binding and receptor block in the rat superior cervical ganglion. Brain Res. 129, 165-168. 3. Carbonetto S. T., Fambrough D. M. and Muller K. J. (1978) Nonequivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons. Proc. Nut1 Acad. Sci., U.S.A. 75, 1016-1020. 4. Changeux J.-P., Devillers-Thiery A. and Chemouilh P. (1984) Acetylcholine receptor: an allosteric protein. Science 225, 1335-1345. 5. Chiappinelli V. A. (1983) Kappa-bungarotoxin: a probe for the neuronal nicotinic receptor in the avian ciliary ganglion. Brain Res. 277, 9-22. Sa. Chiappinelh V. A. (In press) Actions of snake venom toxins on neuronal nicotinic receptors and other neuronal receptors. Pharmacol. Thw. 6. Chiappinelli V. A., Cohen J. B. and Zigmond R. E. (1981) The effects of a- and p-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia. Bruin Res. 211, 1077126. 7. Chiappinelli V. A. and Dryer S. E. (1984) Nicotinic transmission in sympathetic ganglia: blockade by the snake venom neurotoxin kappa-bungarotoxin. Neurosci. Left. SO, 239-244. 8. Chiappinelli V. A., Wolf K. and Ciarleglio A. (1985) Kappa-bungarotoxin: binding of a neuronal nicotinic receptor probe to chick optic lobe and skeletal muscle. Sot. Neurosci. Abs. 11, 92. 9. Chiappinelli V. A., Wolf K., DeBin J. and Holt 1. L. (In press) Kappa-flavitoxin: isolation of a new neuronal nicotinic receptor antagonist that is structurally related to kappa-bungarotoxin. Brain Res. 10. Cohen J. B., Weber M. and Changeux J.-P. (1974) Effects of local anesthetics and calcium on the interaction of cholinergic ligands with the nicotinic receptor protein from Torpedo marmorata. Molec. Pharmac. 10, -932. 11. Culver P., Burch M., Potenza C., Wasserman L., Fenical W. and Taylor P. (1985) Structure-activity relationships for the irreversible blockade of nicotinic receptor agonist sites by lophotoxin and congeneric diterpene lactones. Molec. Pharmac. 28, 43&444. 12. Culver P., Fenical W. and Taylor P. (1984) Lophotoxin irreversibly inactivates the nicotinic acetylchohne receptor by preferential association at one of the two primary agonist sites. J. biol. Chem. 259, 3763-3770. 13. Culver P. and Jacobs R. S. (1981) Lophotoxin: A neuromuscular acting toxin from the sea whip (Lophogorgiu rigidu). Toxicon 19, 825-830. 14. Dryer S. E. and Chiappinelli V. A. (1983) Kappa-bungarotoxin: An intracellular study demonstrating blockade of neuronal nicotinic receptors by a snake neurotoxin. Bruin Res. 289, 317-321. 15. Dryer S. E. and Chiappinelli V. A. (1985a) An intracellular study of synaptic transmission and dendritic morphology in sympathetic neurons of the chick embryo. Deu. Brain Res. 22, 99-111. 16. Dryer S. E. and Chiappinelli V. A. (1985b) Properties of choroid and cihary neurons in the avian ciliary ganglion and evidence for substance P as a neurotransmitter. J. Neurosci. 5, 26542661. 17. Fenical W., Okuda R. K., Bandurraga M. W., Culver P. and Jacobs R. S. (1981) Lophotoxin: A novel neuromuscular toxin from Pacific sea whips of the genus Lophogorgia. Science 212, 1512-1514. 18. Freeman J. A., Schmidt J. T. and Oswald R. E. (1980) Effect of a-bungarotoxin on retinotectal synaptic transmission in the goldfish and the toad. Neuroscience 5, 929-942. 19. Grant G. A. and Chiappinelli V. A. (1985) Kappa-bungarotoxin: complete amino acid sequence of a neuronal nicotinic receptor probe. Biochemistry 24, 1532-1537. 20. Karlin A. (1980) Molecular properties of nicotinic acetylcholine receptors. In The Cell Surface and Neuronal Function (eds Cotman C. W., Poste G. and Nicolson G. L.), pp. 191-260. Elsevier, New York. 21. Karlsson E. (1979) Chemistry of protein toxins in snake venoms. In Snake Venoms, Hundb. Exp. Pharm. (ed. Lee C. Y.), Vol 52, pp. 159-212. Springer, Berlin. 22. Krodel E. K., Beckman R. A. and Cohen J. B. (1979) Identification of a local anesthetic binding site in nicotinic postsynaptic membranes isolated from Torpedo marmorata electric tissue. Molec. Pharmac. 15, 294-3 12. 23. Langdon R. B. and Jacobs R. S. (1983) Quanta1 analysis indicates an a-toxin-like block by lophotoxin, a non-ionic marine natural product. Life Sci. 32, 1223-1228. 24. Langdon R. B. and Jacobs R. S. (1985) Irreversible autonomic actions by lophotoxin suggest utility as a probe for both C6 and Cl0 nicotinic receptors. Bruin Res. 359, 233-238. 25. Loring R. H., Chiappinelli V. A., Zigmond R. E. and Cohen J. B. (1984) Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion. Neuroscience 11, 989-999. 26. Loring R. H., Dahm L. M. and Zigmond R. E. (1985) Localization of alpha-bungarotoxin binding sites in the cihary ganglion of the embryonic chick: An autoradiographic study at the light and electron microscopic level. Neuroscience 14, 6454i60. 27. Loring R. H. and Zigmond R. E. (1985) Amino acid sequence of a neurotoxin that blocks neuronal nicotinic receptors and the localization of its binding sites in chick ciliary ganglion. Sot. Neurosci. Abs. 11, 92. 28. Marshall L. M. (1981) Synaptic localization of a-bungarotoxin binding which blocks nicotinic transmission at frog sympathetic neurons. Proc. Null Acad. Sci., U.S.A. 78, 1948-1952. 29. Martin A. R. and Pilar G. (1963) Dual mode of synaptic transmission in the avian cihary ganglion. J. Physiol. 168, 443-463. 30. Marwitt G., Pilar G. and Weakly J. N. (1971) Characterization of two ganglion cell populations in avian ciliary ganglia. Brain Res. 25. 317-334.
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bf.
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et ai.
31. Matsumoto M., Riker W. K., Takashima K., Goss J. R. and Mela-Riker L. (1985) DMSO effects on synaptic facilitation and calcium dependence in bullfrog sympathetic ganglion. Eur. J. Pharmac. 109, 213-218. 32. McEachern A. E., Margiotta J. F. and Berg D. K. (1985) Gamma-aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture. J. Neurosci. 5, 269&2695. 33. Neubig R. R. and Cohen J. B. (1979) Equilibrium binding of )H-tubocurarine and ‘H-acetylcholine by Torpedo postsynaptic membranes: Stoichiometry and ligand interactions. Biochemistry 18, 54645475. 34. Ogden D. C., Siegelbaum S. A. and Colquhoun D. (1981) Block ofacetylcholine-activated ion channels by an uncharged local anesthetic. Nature 289, 596-598. 35. Ravdin P. M. and Berg D. K. (1979) Inhibition of neuronal acetylcholine sensitivity by cc-toxins from Bungurus multicinctus venom. Proc. Nat1 Acad. Sci., U.S.A. 76, 2072-2076. 36. Ravdin P. M., Nitkin R. M. and Berg D. K. (1981) Internalization of a-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity. J. Neurosci. 1, 849-861. 37. Sine S. M. and Taylor P. (1981) Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J. biol. Chem. 256, 6692-6699. 38. Taylor P. (1985) Ganglionic stimulating and blocking agents. In The Pharmacological Basis of Therapeutics (eds Gillman A. G., Goodman L. S., Rall T. W. and Murad F.), pp. 215-221. Macmillan, New York. (Accepted 17 June 1986)
03~~522/87 %3.00+ 0.00 Pergamon Journals Ltd 0 1987 IBRO
LOP~OTOXIN: SELECTIVE NICOTINIC TRANSMISSION GANGLIA BY A CORAL
BLOCKADE OF IN AUTONOMIC NEUROTOXIN
EVA M. SORENSON,* PAUL CULVER? and VINCENT A. CHIAPPINELLI*$ *Department of Pha~acology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, U.S.A.; TDivision of Pharmacology, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A. Abstract-Lophotoxin is a diterpene lactone isolated from gorgonian corals. The toxin has previously been shown to bind with high affinity to an acetylcholine recognition site located on skeletal muscle nicotinic receptors, producing an essentially irreversible blockade of neuromuscular transmission. Lophotoxin has also been shown to block nicotinic transmission in autonomic ganglia of the frog and in ileal strips of guinea pig and rabbit. The effects of lophotoxin have now been examined on neuronal nicotinic receptors in autonomic ganglia of the chick and rat. Low concentrations of lophotoxin (1 PM) produce a blockade of neuronal nicotinic transmission which is partially reversed by 3-5 h of washing out the toxin. The blockade produced by higher concentrations of lophotoxin (up to 32 PM) is not reversed during a similar washout period. Prior exposure to d-tubocurarine, a competitive nicotinic antagonist, can partially protect ganglia against exposure to lophotoxin. In contrast the local anesthetic QX-314, a noncompetitive nicotinic antagonist, does not protect ganglia against lophotoxin exposure. Lophotoxin binds to a site in ganglia identified by [lzsI]K-bungarotoxin which appears to be on the neuronal nicotinic receptor. Intracellular recordings reveal that lophotoxin has no effect on either muscarinic responses or on responses to y-aminobutyrate in autonomic ganglia. Passive and active membrane properties of the neurons are unaffected by lophotoxin except for the blockade of nicotinic responses. It is concluded that lophotoxin is a selective, high-affinity antagonist at the neuronal nicotinic receptor. The long-term nature of the blockade with lophotoxin suggests that the toxin will be of considerable value as a probe for characterizing the ganglionic nicotinic receptor.
Nicotinic acetylcholine receptors are present in vertebrate nervous tissue in both autonomic ganglia and the central nervous system. These neuronal nicotinic receptors share some characteristics with the nicotinic receptors found in skeletal muscle and in the muscle-
derived electric organs of certain electric fish. While nicotine is a potent antagonist of acetylcholine action at nicotinic receptors in both ganglia and muscle, other drugs can be used to differentiate these two receptor subtypes. For example, trimethaphan and hexamethonium are potent inhibitors of neuronal nicotinic receptors, but are active at muscle receptors o&y at much higher concentrations.38 The most important ligands for characterizing muscle and muscle-derived nicotinic receptors have been the cc-neurotoxins isolated from the venom of various elapid and hydrophid snakes.2’ These basic poly~ptides (Mr = 65~-gOO0) bind with extremely high affinity (& = 10-9-10-‘2 M) and specificity to the acetylcholine recognition sites on muscle receptors.4.20a-Bungarotoxin, purified from the venom of Bungarus multicinctus, is one of the most potent of these E-neurotoxins in blocking muscle receptors. Yet most a-neurotoxins, including tr-bungarotoxin, demonstrate little or no physiological activity at a number of neuronal nicotinic receptors2.3,6 (but see $Author to whom correspondence should be addressed. Abbreviation: GABA, y-aminobutyrate.
also Refs 18 and 28). A related family of snake toxins, termed the K-neurotoxins, block neuronal nicotinic receptors at doses well below those required for blockade of muscle receptors.5a**The characteristics of the blockade support the conclusion that Ic-neurotoxins bind with high affinity to acetylcholine recognition sites on neuronal nicotinic receptors.s~7~‘4 Radiolabeled derivatives of Fc-neurotoxins, such as [1251]K-bungarotoxin, can thus be used as probes for the neuronal nicotinic receptors5 Few other ligands are available which can be similarly used. Recently the structure of lophotoxin, a diterpene lactone isolated from gorgonian corals, has been described.” At doses of 2-32 PM, this toxin produces an essentially irreversible blockade of nicotinic neuromuscular transmission.‘.‘3,23 Intracellular electrophysiological studies demonstrate that lophotoxin blocks miniature endplate potentials, endplate potentials, and the direct actions of acetylcholine in skeletal muscle cells while having very little or no effect on prejunctional nerve terminals.‘.23 Binding studies reveal that lophotoxin blocks transmission by binding to the acetylcholine recognition sites on muscle nicotinic receptors.‘* At concentrations of 1632 p M, lophotoxin also blocks nicotinic transmission in frog sympathetic ganglia and rabbit and guinea pig ileal strips in an apparently irreversible manner.24 The present study was undertaken to further char875
EVA M. SORENSON et al.
876
Control
Response
Ciliary Ganglion
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Fig. I. Effect of lophotoxin on transmission in the chick ciliary ganglion. Electrical stimulation of the preganglionic nerve (break in record) results in three separate compound action potentials, recorded extracellularly from the postsynaptic ciliary nerve. The response with the shortest latency (arrow) represents ciliary neurons activated by electrical coupling potentials. The largest response is due to ciliary neurons activated chemically through nicotinic receptors. The small response with the longest latency represents choroid neurons activated by nicotinic receptors. Exposure to I% (v/v) dimethyl sulfoxide (DMSO) had no detectable effect on the cells. The flow was stopped during this 60 min incubation with 1% dimethyl sulfoxide, as it was during exposures to toxins. Exposure to 32 p M lophotoxin completely blocked nicotinic transmission in both ciliary and choroid cells in 30 min. This effect was not reversible over the time period examined, which ranged up to 5 h after washing out the toxin. Note that lophotoxin had no effect at any time on electrically mediated synaptic transmission. Calibration bars: 1 mV and 2 ms.
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EXPERIMENTAL PROCEDURES Methods for intracellular”” and extracellularS recording from isolated, intact autonomic ganglia have previously been described. Briefly, for extracellular recording, suction electrodes were used to stimulate preganglionic nerve trunks at 1Hz and record the resulting compound action
potentials in postganglionic nerve trunks. Responses were differentially amplified (Tektronix 5A22N) and displayed on an oscilloscope. Intracellular recordings were made by standard bridge-balance methods using a WPI-M707 electrometer. Glass microelectrodes pulled on an Industrial Science Associates (Ridgewood, New York) electrode puller were filled with 3 M KC1 and had resistances of 60-120 MD. Ciliary ganglia and lumbar sympathetic chain ganglia were dissected from 18-20 days of incubation White Leghorn chick embryos (SPAFAS, Peoria, IL). Superior cervical ganglia were removed from 150-200 g SpragueDawley rats. All ganglia were desheathed prior to electrophysiological recordings, which were performed either at 22°C (extracellular recording) or at 35°C (intracellular recording). The physiological saline used was of the following composition: NaCl (150mM); KC1 (3 mM); CaCl, (5 mM); MgCI, (2 mM); glucose (17 mM); HEPES (IO mM); pH 7.4, bubbled with 100% 0,. The ganglia were perfused with physiological saline at 24 ml/min. The flow was stopped during exposures to lophotoxin and h--bungarotoxin in order to conserve the toxins. Control experiments indicated that 60-90min periods of stopped flow in toxin-free physiological saline had no effect on ganglia neurons (see Fig. 1). Pressure injections of carbachol and GABA were performed with a small pressure pipette placed 3 mm “upstream” from the ganglion.‘4 IO ~1
Lophotoxin blocks neuronal nicotinic transmission of drug (lOa M) dissolved in physiological saline was injected while maintaining the 2-4 mi/min flow rate in the recording chamber, which had a total capacity of 1.7mi. Binding experiments were performed at 22°C on freshly dissected whole chick ciiiary ganglia as previously described.5 Ganglia were placed into plastic microwells with fine forceps. The incubation buffer (final volume = 30 ~1) was identical to the physiological saline described above, with the addition of 1mg/mi bovine serum albumin. Preincubations with various concentrations of drugs were for 1 h, followed by a 2 h incubation with added [Y]Kbungarotoxin (final concentration = 20 nM). At the end of this incubation period, ganglia were washed with several rinses of 4°C incubation buffer for a total of 10min. Individual ganglia were then placed in vials and bound radioactivity was determined in a y-counter. Control experiments demonstrated that 1% (v/v) dimethyl sulfoxide had no effect on [‘251]K-bungarotoxin binding. Materials Crystalline iophotoxin was prepared according to the method of Fenicai et al.” Due to the hydrophobic nature of the toxin, stock solutions of IOmM lophotoxin were prepared in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide to which ganglia were exposed never exceeded 1% (v/v). Control experiments (see Fig. 1) indicated that 1% dimethyl sulfoxide had no effect on the physiological properties of ganglia neurons. In a recent study, similar concentrations of dimethyl suifoxide did not interfere with transmission in bullfrog sympathetic ganglia, although higher concentrations (3-10%) produced a facilitatory effect on transmission.)’ K-Bungarotoxin and a-bungarotoxin were purified from the crude venom of Bungarus multicinctus(Miami Serpentarium, Salt Lake City, UT, Lot No. BM&lOSTLZ) as reported.’ QX-314 and QX-222 were the generous gifts of Astra Lakemedel AB, Sodertalje, Sweden. Other drugs and chemicals were obtained from Sigma Chemicals, St. Louis, MO. RESULTS
Blockade of nicotinic transmission by lophotoxin In isolated, intact ciliary ganglia, electrical stimulation of the preganglionic oculomotor nerve elicited up to three distinct compound action potentials, recorded extracelluiariy from the postsynaptic ciliary nerves (Fig. 1). The shortest latency response was due to ciliary neurons which were activated by electrical coupling potentials. 29 The largest response represented ciliary neurons activated chemically via nicotinic acetylcholine receptors. In some ganglia, a third peak with a longer latency was observed and was due to choroid neurons activated by nicotinic receptors.30 At a dose of 32 PM, lophotoxin completely blocked nicotinic transmission in 30min in both ciliary and choroid neurons (Fig. I). Following exposure to 32 ,UM iophotoxin, no recovery was seen in nicotinic transmission in the ganglia for up to 5 h after washing out the toxin (Fig. 1). The compound action potential representing ciliary neurons activated by electrical synaptic transmission was unaffected by iophotoxin. Lower doses of lophotoxin required considerably longer periods of exposure to obtain a complete blockade. The lowest dose to achieve a complete block of nicotinic transmission in 90 min was 1 FM lophotoxin (Fig. 2). Following exposure to low doses
877
of lophotoxin, a partial recovery of nicotinic transmission was seen (Fig. 2). This recovery required at least 3 h of washing out of lophotoxin. Lophotoxin also blocked nicotinic transmission in the rat superior cervical ganglion, although higher doses of lophotoxin were required for a complete blockade in the rat. For example, after 30min at 3 PM lophotoxin, nicotinic transmission in chick ganglia was completely blocked, while rat ganglia were totally unaffected. A dose of 32 PM iophotoxin did completely block nicotinic transmission in rat ganglia after an exposure of 1 h. Protection experiments
Previous studies have demonstrated that the reversible competitive antagonist d-tubocurarine can be used to protect autonomic ganglia against the long-term blockade produced by ~-bungarotoxin.’ A similar experiment was carried out with lophotoxin. Pre-exposure of ciliary ganglia to 300 p M d-tubocurarine partially protected against the subsequent addition of 3 PM lophotoxin (Fig. 3). Virtually identical results were obtained in two different ganglia using 3OOpM d-tubocura~ne. This dose of d-tubocurarine was approximately &fold higher than the concentration required for a complete blockade of nicotinic transmission in the ganglia. A ten-fold lower dose of d-tubocurarine (30pM) did not protect against subsequent exposure to 3 PM lophotoxin in another ganglion. Doses of d-tubocurarine higher than 300,uM were not tried due to the possible nonselective effects of the drug at higher concentrations. Local anesthetics have been shown to block the actions of acetylcholine at nicotinic receptors in a noncom~titive fashion. lo These compounds appear to block the ion channel associated with the nicotinic receptor. 22,34Two lidocaine derivatives were selected to determine whether local anesthetics could protect against the long-term blockade caused by lophotoxin. The use of the quaternary lidocaine derivatives, QX3 14 (quaternary N-ethyl) and QX-222 (quaternary ~-methyl), kept nonspecific absorption of drug into ceil membranes at a minimum, providing rapid washout times. QX-314 produced a complete blockade of nicotinic transmission at 100 PM in chick ciliary ganglia and was approximately three-fold more potent than QX-222. Complete recovery from QX314 exposure required a washout period of between 10 and 40 min. As with the d-tubocurarine protection experiments, the QX-314 concentration was varied between the minimum required for a blockade (100 PM) and an 8-fold higher dose in a series of protection experiments in four separate ganglia. Axonal conduction was not impaired by these concentrations of QX-314, since electrically mediated ciliary neuron responses remained intact (Fig, 4B). Even at the highest dose tested (800 PM) QX-314 did not protect ganglia against the subsequent addition of 3 PM iophotoxin, as after a 3 h washout of both
EVA M. SORENSON et al.
878
Control Ciliary
Response
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Fig. 2. Lophotoxin blockade is partially reversible. Preganglionic stimulation elicits electrically mediated ciliary neuron response (arrows; partially obscured by stimulation artifacts), chemically mediated ciliary neuron response (largest response) and chemically mediated choroid neuron response. Both nicotinic responses are completely blocked after exposure to I PM lophotoxin for 90 min, while the electrical response is unaffected. Washout of the toxin began after 90 min exposure. Recovery of the chemically mediated responses began to appear at 3 h of washout, and by 5.3 h of washout, both ciliary and choroid responses were recognizable. Calibration bars: 0.5 mV and 2 ms.
toxins no nicotinic transmission could be detected (Fig. 4A). Similarly, pre-exposure of ganglia to QX314 at either 100pM or 800pM was unable to protect against the subsequent addition of 100nM K-bungarotoxin (Fig. 4B).
Intracellular
recording
Intracellular recording was used to further examine the nature of the blockade seen with lophotoxin. In these experiments, chick lumbar sympathetic chain ganglia were continuously perfused with physiological saline containing 1.5 PM atropine. This dose of atropine was sufficient to completely block muscarinic responses in the ganglia, while leaving nicotinic responses intact.’ Under control conditions in the presence of atropine, carbachol produced a robust depolarization associated with a decrease in input resistance (Fig. 5A). A similar response was seen following exposure to GABA (Fig. 5A). The
GABA response was mediated by a bicuculline- and picrotoxin-sensitive GABA, receptor.j2 Following exposure to lophotoxin, the nicotinic response to carbachol was almost completely blocked, while the GABA response was unchanged (Fig. 5B). After lophotoxin exposure, neurons exhibited normal resting membrane potentials and displayed overshooting action potentials in response to direct stimulation through the intracellular electrode (Fig. 5B). Similar results were obtained from a total of 11 neurons after lophotoxin exposure in three different sympathetic ganglia preparations. To determine whether muscarinic responses were affected by exposure to lophotoxin, atropine was deleted from the physiological saline. Under these conditions, the muscarinic response to carbachol (a depolarization with an increase in input resistance15) persisted after lophotoxin exposure (Fig. SC). Similar results were seen in three different cells from two different preparations.
Lophotoxin blocks neuronal nicotinic transmission
Control Response
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Fig. 3. d-Tubocurarine protects against lophotoxin blockade. When exposed to 300 PM d-tubocurarine for 50 min, chemical transmission in ciliary neurons is completely blocked in 5 mitt, and complete recovery requires 20 min of washout (not shown). The protection experiment consists of a 10 min exposure to d-tubocurarine, followed by a 40 min exposure to d-tubocurarine and 3 PM lophotoxin. Washout is for Smin with 3OOgM d-tubocurarine, followed by continued washout with normal physiological saline. Partial recovery from exposure to both toxins is seen after 16 min, and the recovery is slightly increased in size by 2.4 h of washout. Finally, 3 FM lophotoxin is applied to the ganglion for 25 min. A complete block of the remaining response occurs in II min, and no recovery is seen after 2 h of washing out the toxin. Calibration bars: 0.2 mV and 2 ms. Binding experiments
was blocked by IO PM lophotoxin, while 30~1cM lophotoxin blocked 99% of this specific binding [‘*‘I]~-Bungarotoxin binds to two different phar(Fig. 6). Similar results were obtained in three differmacologically nicotinic sites in chick ciliary ganglia.5 Most (84%) of the specific binding of [1251]rc- ent binding experiments. Thus, lophotoxin and ?c-bungarotoxin bind to the same nicotinic sites in bungarotoxin is to a site which is also detected ciliary ganglia, a portion of which are associated with by the physiologically inactive a-bungarotoxin the neuronal nicotinic receptor. (Fig. 6). Considerable electrophysiologicals~6~‘4~2s and anatomical26 evidence indicates that this neurotoxin DISCUSSION binding site is not associated with ganglionic nicotinic transmission, The remaining 16% of specific The present results support the conclusion that [‘251]?c-bungarotoxin binding is not blocked by 1 PM lophotoxin blocks nicotinic transmission in autox-bungarotoxin (Fig. 6) but is blocked by the nico- nomic ganglia by binding to acetylcholine recognition tinic receptor affinity agent bromoacetylcholine.5 This sites on neuronal nicotinic receptors. An identical binding site appears to be located on the physio- mechanism has previously been proposed to account logically detected neuronal nicotinic receptor.5.25~“7 To for lophotoxin’s action at the neuromuscular juncdetermine whether lophotoxin binds to either of these tion.‘,‘2,23The selectivity of lophotoxin for nicotinic [‘251]~-bungarotoxin sites, ganglia were pre-exposed receptors was demonstrated by its inability to block to two different concentrations of lophotoxin. Most muscarinic or GABAergic receptors in autonomic of the ]‘251]rc-bungarotoxin specific binding (85%) ganglia.
880
EVA M. SORENSON et al
A Control Ciliary
800
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Response Ganglion
800
B Control Response Ciliary Ganglion
+
pM QX 314
100 nM K-Bungarotoxin
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3.2 hr
Wash 2.0 hr
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Fig. 4. Lidocaine derivative QX-314 does not protect against lophotoxin blockade. Protocol for protection experiment is identical to that described for d-tubocurarine protection experiment (legend to Fig. 3) except that 800nM QX-314 is substituted for d-tubocurarine. Complete recovery from exposure to 800 PM QX-314 alone required 40 min of washout (not shown). (A) 800 PM QX-314 does not protect against exposure to 3 PM lophotoxin, since no recovery is seen after 2.2 h and 3.2 h of washing out the toxin. (B) 800pM QX-314 does not protect against exposure to 100 nM h--bungarotoxin. Note that the electrically mediated ciliary neuron response (arrow) is unaffected by either QX-314 or h--bungarotoxin. The recovery from K-bungarotoxin exposure begins at 3.3 h of washout, which is similar to recovery times following exposure to x-bungarotoxin alone.s Calibration bars: (A) 0.2 mV and 2 ms; (B) 0.5 mV and 2 ms. Extracellular electrophysiology A single study has previously characterized the effects of lophotoxin in ganglionic preparations.24 At a high dose (1632 p M), lophotoxin blocked nicotinic responses recorded extracellularly in frog sympathetic ganglia and in ileal strips from rabbit and guinea pig. In the frog ganglia, no recovery was seen after 16 h of washing out the toxin. The effects of lower doses of lophotoxin were not examined. No protection experiments were undertaken in this study, nor were any intracellular recordings made. In the present study, extracellular recordings from chick and rat autonomic ganglia revealed an apparently irreversible blockade of nicotinic transmission at high concentrations (32 p M) of lophotoxin. At lower doses (1 PM) of the toxin, however, the blockade was at least partially reversible after 3-5 h of washing out the toxin. Thus, it is not clear whether any effects of lophotoxin in ganglia are in fact “irreversible”. Given sufficient time, the ganglia might also have recovered from the higher doses of the toxin. Lophotoxin produces an apparently irreversible blockade of nicotinic transmission in a variety of in neuromuscular preparations.‘~‘2~“~23~24 Results BC3H-1 cells (a muscle cell line) are consistent with lophotoxin binding in a two-stage process to the muscle nicotinic receptor. ‘* The initial binding is of relatively low affinity and is reversible. However, with continued exposure to the toxin, the binding proceeds to an apparently irreversible stage. Such a process
may explain why recovery ganglia only at low doses transition to irreversible occurred by the end of the
was seen in autonomic of lophotoxin, where a binding may not have exposure period.
Protection experiments The long-term blockade of nicotinic transmission produced in chick ganglia by 3 FM lophotoxin could be partially prevented by prior exposure of ganglia to the competitive nicotinic antagonist d-tubocurarine. d-Tubocurarine binds selectively to acetylcholine recognition sites on nicotinic receptors.” At the highest dose of d-tubocurarine used (300pM), approximately 40% of the nicotinic response was protected. Lower doses (30 PM) were not effective in protecting against lophotoxin. The reasons that relatively high doses of d-tubocurarine were required and only a partial protection was seen are not known, although the slow off-rate of lophotoxin likely contributed to this difficulty in protecting against the toxin. In addition, lophotoxin is strongly lipophilic, and during the washout period, toxin molecules dissolved in ganglion cell membranes may diffuse and become associated with nicotinic receptors at a time when d-tubocurarine is no longer present in solution. Evidence for such a phenomenon has been reported in intact BC3H-1 cells.‘* The protection against lophotoxin afforded by d-tubocurarine is due to the two toxins interacting at identical sites, presumably acetylcholine recognition sites on neuronal nicotinic receptors, Local anes-
Lophotoxin
blocks
A. Control Responses
neuronal
nicotinic
881
transmission
in 1.5 FM Atropine
Carb
GABA
B. Responses After Lophotoxin Carb
Exposure
in 1.5 PM Atropine
GABA
50
mv 0.5
0.7
“AI
__
min
-
1
C. Muscarinic
Response
Unaffected
Carb
GABA
+
$
by Lophotoxin
0.5
min
Fig. 5. Intracellular records from chick lumbar sympathetic ganglia. (A) In the presence of 1.5pM atropine, pressure injection of carbachol (Carb; 10~1 of 10e2 M) produces a depolarization in a sympathetic cell. This depolarization is associated with a decrease in input resistance, measured as the voltage response to constant pulses of hyperpolarizing current. y-Aminobutyrate (GABA; 10~1 of 10-2M) also produces a depolarization with a decrease in input resistance. (B) The ganglia are subsequently exposed to 32 PM lophotoxin for 20 min. Approximately 30 min after lophotoxin exposure, the response to carbachol (10 yl of 10e2 M) in another cell is almost completely blocked. Response to GABA (IO ~1 of 10m2 M) is similar to that seen before lophotoxin exposure. A depolarizing current pulse delivered after recovery from GABA (asterisk) is also shown on a greatly expanded time scale (lower record). The cell exhibits normal-appearing, overshooting action potentials in response to the depolarizing current pulse. (C) To determine whether lophotoxin has any effect on muscarinic responses in sympathetic ganglia, atropine was deleted from the physiological saline in another preparation. Following exposure to lophotoxin (32 PM for 20 min), the response to carbachol(10 ~1 of 10e2 M) consists of a depolarization associated with an increase in input resistance. This response is muscarinic in nature.15 y-Aminobutyrate (10 ~1 of 10m2 M) produces a depolarization with a decrease in input resistance similar to that seen before lophotoxin exposure. Resting membrane potential for the neurons shown is between -40 mV and - 50 mV.
thetics,
which
are
noncompetitive
nicotinic
antag-
do not bind to these acetylcholine recognition sites,‘0,22,34do not protect chick ganglia from exposure to 3 p M lophotoxin. Similar results have previously been obtained for the muscle nicotinic receptor of BC3H-1 cells.” In this preparation, competitive agonists and antagonists such as carbachol, pancuronium and d-tubocurarine protect against the action of lophotoxin, while the local anesthetic dibucaine is unable to prevent the lophotoxin blockade. onists
that
Intracellular recording Intracellular records from chick autonomic ganglion cells reveal that lophotoxin has a selective effect on postsynaptic neurons. Nicotinic responses to carbachol are almost completely blocked by the toxin at concentrations where muscarinic and GABAergic responses are unaffected. Langdon and Jacobs” report that lophotoxin is also ineffective in blocking muscarinic receptors located in smooth muscle. Thus, the blockade of nicotinic receptors produced by lophotoxin is selective, and is not accompanied by
882
EVA M. SORENSON Edal
q
Total Binding
Lophotoxin
1 FM KBgT 1 pM ABgT
*
Fig. 6, Inhibition of [‘*jI]K-bungarotoxin binding in ciliary ganglia by lophotoxin. Intact ciliary ganglia were pretreated for 1 h with lophotoxin (IOpM or 30,~M), x-bungarotoxin (KBgT: 1 FM), ~-bungarotoxin (ABgT; 1PM), or no toxin (Total Binding), followed by a 2 h incu~tion with 20 nM [1251J~-bungarotoxin. Bars represent mean f S.E.M. (N = 5 in each group). Percentages reported are remaining amount of specific binding (defined as Total Binding minus binding in the presence of 1 pM h--bungarotoxin). *P -C0.025 between binding in the presence of 1PM a-bungarotoxin and binding in the presence of 1 PM K-bungarotoxin.
K-neurotoxins and bromoacetylcholine,’ but is not detected by the inactive a-bungarotoxin. This site appears to be located on the ganglionic nicotinic receptor.s.25+27Lophotoxin inhibits the binding of [‘251]~-bungarotoxin to this selective K-neurotoxin site. inhibition of K-neurotoxin binding Two independent experiments thus confirm that lophotoxin is a competitive antagonist at neuronal Snake venom rc-neurotoxins are potent competitive antagonists at ganghonic nicotinic receptors.” This nicotinic receptors. First, the competitive antagonist class of peptide toxins includes K--bungarotoxin,‘.” d-tubocurarine protects against lophotoxin action. all purified from Bungurus Second, lophotoxin inhibits the binding of [‘251]~Toxin F?’ and Bgt 3. I, 35~36 ~~it~ri~~t~~ venom, and K-flavitoxin’ from the bungarotoxin to a nicotinic site which is located on venom of ~~ngarus Jtaviceps. Preliminary evidence the neuronal nicotinic receptor. Lophotoxin should indicates that the amino acid sequence of Toxin F17 therefore be a useful probe in characterizing neuronal may be identical to the complete amino acid sequence nicotinic receptors. The very slowly reversible or reported for K-bungarotoxin.iY Radiolabeled derivairreversible nature of iophotoxin binding to neuronal tives of ~-bungarotoxin,* Toxin F2’ and rc-ffavitoxin’ nicotinic receptors indicates that radiolabeled lophobind to two nicotinic sites in autonomic ganglia. One toxin may be an important Iigand in the identification of these sites, which is also detected by radiolabeled and purification of this receptor. The high lipid a-bungarotoxin, is not associated with neuronai nico- solubility of lophotoxin may pose problems with high tinic transmission.6 The second site is identified by nonspecific binding. Fortunately, several congeners physiologicaily active compounds such as the of lophotoxin are also active at nicotinic receptors,” any other observable effects on the active or passive membrane properties of the cells. In particular, the cells continue to maintain a normal resting membrane potential and continue to fire action potentials in response to injected depolarizing current.
Lophotoxin suggesting found
for
blocks
neuronal
that a less lipophilic derivative may be binding
experiments.
Acknowledgements-This research was supported by the National Institutes of Health, Research Grant NS 17574 to
nicotinic
transmission
883
V.A.C. We wish to thank Kathleen Wolf for expert technical assistance and preparation of the figures, and Maggie Klevorn for preparation of the manuscript. We are grateful to Dr Bengt Akerman of Astra Lakemedel AB for providing investigational samples of QX-314 and QX-222.
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