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Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles
Camp.
Eiochem.
Physiol.
Vol.
91C.
No.
2. pp.
349-354,
0306~4492/88
1988
ALKALOID TRANSPORT
$3.00
+ 0.00
Pergamon Pressplc
Printed in Great Britain
NEUROTOXINS-DEPENDENT SODIUM IN INSECT SYNAPTIC NERVE-ENDING PARTICLES
A. K. DWIVEDY A.F.R.C. Unit of Insect Neurophysiology and Pharmacology, Dept. of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK (Telephone (0223) 336600) (Received
21 August 1987)
Abstract-l. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplunefa americana) was investigated. of Na+ uptake; 2. Batrachotoxin (K,,, , 0.2 FM) was full agonist as for most effective activator of “Na+ uptake veratridine (K, 5, 2.5 PM) and aconitine (K,,,, 7.6 p M) produced a maximal stimulation that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I, s. I I nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins-stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect svnantosomes than those oreviouslv described for vertebrate brain synaptosomes, cultured neuronal cell, _ nerve membrane vesicles and neuroblastoma cells. 1
variations in the properties of sodium channels. In insects and crustaceans, the access of neurotoxins has been available only to the external surface of axonal preparations. The responses of some neurotoxins are ineffective in cephalopod axons (Romey et al., 1976), though are effective in cockroach and crustacean nerves (Narahashi et al., 1969; Pelhate and Sattelle, 1982). The present investigation describes appropriate conditions for measuring sodium channeldependent **Na+ influx in the synaptosomal fraction isolated from the central nervous system (CNS) of the cockroach, Periplaneta americana. The responses of alkaloid toxins, batrachotoxin (BTX), veratridine (VTN), and aconitine (ACN) which activate specifically to the influx of sodium in insect presynaptic membranes are described. Some of the results have been presented in part elsewhere (Dwivedy, 1986; Dwivedy, 1987).
INTRODUCTION sodium channels play a key role in generation of action potential in many excitable membranes. The detection and modification in the properties of sodium channels have been described with the use of a number of classes of neurotoxins from plant and animal origin (Lazdunski and Renaud, 1982; Catterell, 1986). Neurotoxins are highly specific in their affinity with the receptors of sodium channels and have been defined predominantly as sodium channel blockers (tetrodotoxin, saxitoxin), sodium channel inactivation inhibitors (sea anemone toxin, scorpion toxin) and sodium channel modifiers (batrachotoxin, veratridine, aconitine). The last group of toxins are lipid soluble and have been refereed as alkaloid neurotoxins (Catterall, 1977; Romey and Lazdunski, 1982). Alkaloid neurotoxins, unlike other neurotoxins, affect voltage-sensitive sodium channels simultaneously at the activation, inactivation, ion selectivity and susceptibility of many drugs and toxins (Narahashi, 1982; Naumov, 1983; Khodorov, 1985). Alkaloid neurotoxin-stimulated sodium uptake has been described among a variety of neuronal membrane preparations by measuring **Na+ influx in synaptosomes from rat brain (Krueger and Blaustein, 1980; Tamkun and Catterall, 1981) mouse brain (Ghiasuddin and Soderlund, 1984), cells from mouse neuroblastoma (Catterall, 1977), cultured neurons from rat brain (Couraud et al., 1986), liposomes preparation from rat brain (Talvenheimo et al., 1982) and electroplax of EIectrophorus electricus (Rosenberg et al., 1984). These preparations differ in their response to alkaloid toxins, and further comparative studies are of considerable interest to the knowledge of species-dependent Voltage-sensitive
MATERIALS AND METHODS Chemicals 22NaCI (carrier free, 450mCi/mg) was purchased from Amersham International, UK. Tetrodotoxin (TTX), aconitine (ACN), veratridine (VTD), ouabain, choline chloride, Albumin (fatty acid free bovine serum albumin, BSA), N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES), ethylenediaminetetraacetic acid dipotassium salt (EDTA), ethyleneglycol-bis(-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) and [tris (hydroxymethyl) aminomethane] (Tris) were purchased from S&a Chemicals, UK. Pot&sium cyanide (I&N) was obtained from Fisons Scientific UK. Batrachotoxin (BTX) was a generous gift of Dr John W. Daly, NIH, Bethesda; MD, U.S.A. Deionized and purified reagent grade water was used to prepare all reagent solutions. Preparation of synaptosomes Synaptosomes and abdominal
c 1988 Crown copyright. 349
were prepared from dissected head, thorax ganglion of adult male cockroach, P.
350
A. K.
americana, according to the method as previously described (Dwivedy, 1985, 1987). The insect ganglion were pooled, homogenized in ice-coid 0.25 M sucrose, containing 0.1 M Tris~ch~oride, and 1.0 mM EDTA (pH 7.4). The homogenate was centrifuged (4‘C) at 8OOg for 15 min. The pellet was homogenized twice with the buffered sucrose solution and recentrifuged. The supernatant were combined and centrifuged at 10,SOOg for 20 min. The resultant pellet (P,) was suspended in sucrose buffer (0.1 ml), homogenized gently with 0.25 M sucrose (0.5 ml) containing 13% Ficoll and suspension (0.5 ml) transferred to Beckman uhramicrocentrifuge tubes of 0.7 ml capacity. Buffered sucrose (0.1 ml of 0.2 M sucrose, containing 1.0 mM K-EDTA, pH 7.4) was added gently on top of sucrose- Ficoll mixture, and allowed to equilibrate at 4’C before it was centrifuged (4°C) at 9500g for 60min. The synaptosomal fraction was carefully collected from the interface with micropipette and resuspended in 0.25 M sucrose buffer, and suspension was centrifuged (15,OOOg for 20 min) to obtain the synaptosomes in pellet form. Influx of *2Na+ in the synaptosomes was measured by a slight modification of procedure as described (Dwivedy, 1987). The synaptosomal preparation was suspended in a sodium free low calcium buffer (5 mM glucose, 3 mM MgCl,, 8 mM KCl, 0.5 mM CaCl,, 0.5 mM EGTA, 2 mM ouabain, 2 mM KCN, 20mM Hepes-Tris. pH 7.4). Solutions of appropriate concentrations of toxins and **Na (2mM. 1-2 uCi/ml) both were made in incubation buffer (20 mM choiine ‘chloride, mg/ml BSA, added to suspension buffer used for suspending synaptosomal fraction, pH 7.4). Aliquots of synaptosomal suspension (10 ill, IO-20 pg) were incubated initially with appropriate concentration of toxins for i min in a total volume of 190~1 incubation buffer. The uptake of zzNa+ in synaptosomes was measured after addition of 10~1 of 2zNa+ into the media having a total volume of 200~1 which was equilibrated in a water bath maintained at 21°C. influx measurements were terminated at desired length of time intervals by aspirating off the radioactive assay medium, transferring it onto buffer soaked filter paper (Whatman GF/C) followed by immediate vacuum filtration (Dwivedy, 1987). Since a significant loss of *‘Na+ occurred with the use of various wash medium at different time intervals (Fig. l), each incubation tube and filters were washed twice as rapidly as possible (less than 30 see) with about 1.0 ml of ice cold wash solution (5 mM glucose, 0.8 mM MgSO,, 1.8mM CaCl,, 20 mM choline chloride, 20 mM Hepes-Tris, pH 7.4). Filters were air dried, dissolved in 20% methyl cellosolve, and counted in ACS (Amersham) liquid scintillation (75% counting efficiency). Specific *jNaT uptake was calculated by substracting the non-specitic zzNa+ uptake obtained in presence of TTX and VTD (sodium-channel activator) from that obtained with VTD alone. Measurement of internal r~olumeqf .synuptosomes The intracellular volume of synaptosomes was measured with tritiated PHI water and ~‘4Clinuiin as internal and external volume markers respectively according to the method described by Breer and Knipper (1985). About 80-100 pg of synaptosomal protein -was &tbated for 10 min at 4°C in a reaction medium (0.5 ml) with a mixture of radiolabelled tracer (10 FCi jH,O + “C-carboxyi inulin, 1-2 p Ci/ml). After incubation, the mixture was diluted with 0.5 ml of reaction buffer. It was incubated for 5 min at room temperature, and centrifuged at 10,800g for 5 min in an Eppendorf centrifuge. From each tube, supernatant and residual pellet were separated, both dissolved separately in 0.5 ml of 1% SDS solution and washed with 1.0 ml of buffer before appropriate amount was counted, and internal volume caiculated. The interval volume of synaptosomes was expressed relative to the protein. The protein content of the
DWIVEDY
fraction were measured by the method of Lowry et al. (1951) using crystalline BSA as the standard. RESIJLTS
Eflect of ouabain on f2Na + injh.~
Inhibition of Nat, K+-ATPase by ouabain and synthesis of ATP by KCN both affect the influx of sodium when synaptosomes are exposed to 2mM external sodium concentration. In absence of ouabain (Fig. 2A), there was a relatively low “Na+ influx in synaptosomes (O.lLO.2 nmol/sec/mg of protein). However, with graded increase in the molar concentration of ouabain, the inward movement of **Nat increased and apparently equilibrated at approximately external concentration of 2 x lO-‘M ouabain. The time course of 22Na+ influx in presence of 2 x 10-l M ouabain (Fig. 2B) indicated a linear curve reached between 30-60sec, in contrast to neurobfastoma cells (30-6Omin) when incubated with 5 mM ouabain (Catterail and Nirenberg, 1973). The cell volume measurement suggested synaptosomal intracellular volume about 2.5 pll/mg of protein. Since 22Na+ uptake in presence of 2 x IO-‘M ouabain approached to 0.36 nrno~/se~~rng of synaptosomal protein, the final intracellular concentration of 22Na+ was 0.15 mM to the extracellular concentration of 2.0 mM sodium. The difference between the extents of uptake of ‘*Na+ in presence and absence of ouabain (Fig. 2B) would suggest that synaptosomes maintain an internal Na’ concentration of 0.1-0.2 mM against an externai concentration of 2mM sodium used in the present study. Assay conditions
Effect of sodium concentrations on initial rates of VTD-stimulated (SOpM) specific Z2Na+ uptake is shown in Fig. 3. VTD-stimulated specific 12Na+ uptake was linear only when the sodium concen-
100 aa .!G
9
3
::4 g
rn
80 tb . GO-
.fj
40 -
I a
20 -
O.Ok 0.0
1.0
2.0
3.0
5.0
Time (min)
Fig. 1. Effect of wash medium on the efflux of ‘“Na+. Synaptosomes (15-20 ~8 protein) were incubated in 2 mM 22NaCi (I pCi/ml) at 2 1’ with 50 ,UM veratridine for 30 sec. transferred to filter paper (Whatman GF/C), followed by vacuum filtration, and then rinsed at given length of time intervals in a wash medium consisting of 5 mM glucose. 0.8 mM MgSO,, 1.8 mM CaCI,, 20mM Hepes-Tris, pH 7.4, with either 20 mM NaCl and 8 mM KC1 (O), or 20 mM choline chloride and 8 mM KC1 (0). or 20 mM chofine chloride (O), or 160 (A). The . . mM choline^ chloride . contmuous lme IS a least-square tit to the data
351
Sodium transport in insect synaptosomes (A)
(B)
L4---+-0.0
1o-4
1O-2
10
I Ouabain I ( M )
L00 .
.
.
5.0
.
Time (akin)
Fig. 2. (A) Effect of ouabain concentration on ‘%a+ uptake in the cockroach CNS synaptosomes (O.Ol-0.02mg of protein). Uptake of “Na+ (2mM, IL2~Ci/ml) was measured for 30sec at 2l’C in presence of indicated concentrations of ouabain. (B) Time effect on uptake of j*Na+ in the synaptosomes in absence (V) or presence of 2.0 mM ouabain (0). Each point represents the average of duplicate experiments.
was increased up to 2mM in incubation media. This suggests that insect synaptic membranes have saturable binding sites of sodium channels. Moreover, a higher concentration than 2 mM of sodium in incubation may result increased sodium permeability which could induce depolarization and reduce in electrical driving force for ‘*Na+-influx. The range of protein content for each incubation were about 0.01 S-O.02 mg of synaptosomal protein. Effect of wash medium on the efficacy of “Na+ efflux suggest that a significant loss of sodium would have occurred if wash medium contained 160 mM choline chloride (Fig. 1). A wash medium containing 8 mM KC1 either with 20 mM NaCl or with 20 mM choline chloride effected the rapid loss of sodium. A wash medium consisting of 20mM choline chloride was used maximally for 30 set in each experiment. tration
Time course of sodium channel activation by VTD Sodium channel specific uptake of *‘Na+ in the synaptosomes were determined by subtracting nonspecific “Na+ uptake from VTD-stimulated *?Na+ influx. The initial rate of **Na.+influx in presence of VTD (50 p M) was greatly enhanced than the rate of
non-specific influx of Na+ (in absence of VTD), and deviation in the linearity of specific *‘Nat influx was observed between 30 set to 1 min (Fig. 4). At the same period of incubation, the VTD-stimulated influx of ‘*Nat was inhibited by 1OpM TTX. Since the experimental values for incubation times less than 30 set were highly variable, and there was no appreciable increase in specific influx of ‘*Na-’ after 30 set of incubation, standard incubation time of 30 set was opted for in all subsequent experiments. Sodium channel activation ofBTX, VTD and ACN The alkaloid toxin BTX, VTD and ACN all produced concentration dependent sodium uptake in the synaptosomes (Fig. 5). Values for the toxinconcentration giving a half maximal activation (K,,,) and maximal activation obtained for each toxin are given in Table 1. BTX was a potent stimulator for synaptosomal sodium uptake which produced **Na+ influx increase to 13 and 38 fold higher than that produced for VTD and ACN respectively over nonspecific uptake at maximal activation. VTD and
0.9, 0.8
-
Cl
0
2.0
6.0
4.0
INa+J
tM
x
10.0
IO-~)
Fig. 3. Effect of sodium concentration for veratridinedependent (50pM) *%a+ uptake in the cockroach CNS synaptosomes (0.01-0.02 mg of protein). Uptake of **Na+ was measured for 30 set as described in methods. Symbols represent means + SEM, n = 3 experiments.
I
0.3
k
1.0
I
1
1.5
2.0
I
3.0
Time (min)
Fig. 4. Time courses of 22Na+ uptake in the cockroach CNS synaptosomes (0.01-0.02 mg protein). Non-specific uptake of 22Na+ was measured at 21°C as described under methods in the absence of toxins (a), uptake in presence of 50pM veratridine (e), and in the presence of 50pM veratridine plus IOpM TTX (A). The data points represent the means + SEM (n = 3).
A. K.
352
DWIVEDY DISCUSSION
5
6
I
6
[Activators1
The present result demonstrated that neurotoxins BTX, VTD. and ACN activate the flux of sodium through the voltage dependent sodium channels of insect synaptosomes and are inconsistent with the voltage clamp studies on activation of sodium channels of cockroach giant axons by some alkaloids of plant origins (Pichon, 1976; Pelhate and Sattelle, 1982). Alkaloid neurotoxins induced a steady state of Na+-permeability reactions of the action potential in the insect synaptosomes and the concentration at which TTX inhibited the generation of action potential in insect axon was apparently of same the range that inhibited the “Nat uptake in the cockroach CNS synaptosomes (Fig. 6). However, the movement of “Nat across nerve membrane depends on both the external and internal ion concentrations and the variable factors such as Na ’ K +-ATPase and Na+/Ca + exchange transporter which may influence the sodium channel gating kinetics. Electrophysiological studies have shown that ouahain inhibits the movement of sodium across insect axons though it was insensitive to sodium influx in perineurium and underlying glial cells (Pichon and Treherne, 1974; Schofield and Treherne, 1975). In the present study, a low intracellular steady state of Na +-concentration within synaptic membrane was maintained by the inhibition in the activity of Na + , K +-ATPase. For the measurement of sodium channel-dependent ‘2Na’ influx, insect synaptosomes were routinely incubated in ouabain, KCN and EGTA; and a suitable washing procedure for incubated synaptosomes was followed. A low concentration of sodium for incubation was determined so that membrane potential is unaffected by the increased sodium permeability caused by the neurotoxin treatment. Appropriate experimental conditions for incubation of synaptosomes resulted in initial rates of non-specific “Nat flux considerably lower than the values obtained using otherwise similar conditions in German cockroach nerve membrane preparation (Rashatwar and Matsumura. 1985).
3
4
(-10 log MI
Fig. 5. Concentration--effect curves for alkaloids toxin dependent 22Na+ uptake (difference between the uptake in presence and absence of toxins) in the cockroach CNS synaptosomes (O.Ol--0.02mg of protein). Uptake was measured at indicated concentrations. Symbols represent the means 2 SEM. n = 4, BTX (a). VTD (I). ACN (A).
ACN both were less effective giving 71% and 43% of the activation observed for BTX at maximally effective concentrations (Table I). 2zNa + uptake by TTX
Inhibition of sped@
The VTD-stimulated (50 p M) uptake of *‘Na + was inhibited by TTX in a concentration dependent manner (Fig. 6). The apparent IC,, for TTX was about 11 nM, and a higher concentration above IO !itM of TTX completely blocked VTD-stimulated “Na + influx.
Table
I.
Comparison
vesicles.
cultured
of the activakw
mouse
neuronal
of sodium
cella.
and
channels
mouse
of vertebrate
neuroblastoma
brain
cells
bv
synaptosomes.
BTX.
VTD
brain
and
membrane
ACN.
ND.
not
Actrvators BTX
VTD
CNS synaptosomes
Insect
(present Mouse
brain
synaptosomes:
brain
synaptosomes$
Rat
brain
synaptosomes~
Cultured 112.
neuroblastomaq mouse i3-dav
neuronal
fetal
*Concentration activation $Data
from
activation of Ghiasuddin
cell**
giving
a
equation available
(100)t
2.5
0 5 (71)
75
$,
SCtiVBllOll
c
(I 7 (43)
IlData
of Krueger
‘IData
of Catterall of Courdud
0.4
8.0 (100)
34.5
5.9 (73)
19.6
3.7 (46)
0.5
0.9 (100)
13.0
0.5 (55)
14.0
0.2 (22)
3.0
0.6 (50)
0.5
1.2(100)
0.7
0.9 (100)
29.0
0.1 (II)
0.2
132.7 (100)
10.4
67.3 (51)
ND 3.6
0.02 (2) ND
half-maximal according
(nmol/sec/mg and
of protein).
Soderlund
and Blaustein (1977).
to
activation. Wilkinson
A (1961)
basic
computer
in evalualing
programme the
values
for
was
designed for
&,,
and
maximai
data.
gData of Tamkun and Catterall
**Data
0.7
activation
brain1
(PM)
Michaelis-Menten tMaximal
0 2
Maximal
K/bi
study)
Rat
Mouse
actwation
..-__K,, <*
______-..
ACN
MaxImal
Maximal
lnmtal maximal
(1981).
maximal
(1980).
maximal
maximal
et al. (1986).
(1984).
activation maximal
rate m absence activation
activation activation
of activator
(nmol/min!mg
(nmol:min/mg
of protein).
(nmol!min/mg
of protein).
(nmol/min/culture).
activation
was 0.1 nmol:sec;mg of protein).
(nmol/min:lO*
cells).
of protein.
353
Sodium transport in insect synaptosomes
-100
-75
- 50
-25
p
B. @ E 4 6 ,x 3
-0
[TTX]
(610losM)
Fig. 6. The effect of different concentrations of tetrodotoxin on the veratridine induced (50 PM) **Na+ uptake in the synaptosomes (O.OlLO.02 mg of protein). The indicated concentrations of tetrodotoxins were added, and **Na+ uptake was measured as described. The data are presented as the means k SEM, n = 4.
The present study CNS synaptosomes
on
sodium
transport
in insect
demonstrates a concentration dependence of alkaloid neurotoxins for the activation of voltage sensitive sodium channels-ionophore. The potency of BTX-stimulated sodium transport was greater (about ten times that of VTD) and similar to the magnitude of that observed for the synaptosomes from the brain of rat and mouse (Table 1). The VTD-stimulated sodium uptake in the synaptosomes from rat cortex, rat cerebellum and mouse brain was found to be 2.6, 1.2 and 1.9 nmol/mg of protein/set respectively and the concentration giving a half maximal activation for both BTX and VTDstimulated influx of **Na+ was found to be dependent on temperature-giving a lower value at the higher temperature (Harris and Bruno, 1984). The other factors likely to contribute to the differences in the rate of specific *‘Na+-flux may include different sodium concentration used and differences in the composition of loading media. Neurotoxins affect the kinetics of sodium channel acivation during membrane depolarization as suggested by a shift in the maximum sodium conductance in nerve membranes (Grischenko et al., 1983; Khodorov, 1985). BTX interacts preferentially with open channels and their modification is fairly stable, while that by VTD is not stable. and ACN pronounced inactivation of ACN-modified sodium channels provides the reason for the fact that ACN produced much smaller increases in “Nat-flux in synaptosomes than did VTD and BTX. The activation of sodium channels of cockroach CNS synaptosomes by BTX, VTD and ACN is compared to that of vertebrate brain synaptosomes, cultured neuronal cells, and neuroblastoma cells in Table I. It indicates the differences in the potency of neurotoxins, BTX being a full agonist, VTD and ACN both partial agonists in insect CNS synaptosomes similar to the observations in other nerve membrane preparations. However, the neurotoxins produced characteristic different responses in sodium channels of insect nerve membrane preparations for both to the values of K, 5 and the extent of maximal activatton. In particular,
the partial agonists VTD and ACN had a greater intrinsic activity in activating sodium channel to 71% and 41% of BTX-maximal activator as compared with SO-%% and 2-22% in rat brain synaptosomes and 5 I % and 2% in neuroblastoma cells. The study suggests that insect synaptosomes, a subcellular membrane preparation of CNS of Peripluneta americana, retain functional properties that are identical with those of intact nerve cells, and provide an ideal subcellular preparation for studying the functional and structural properties of nerve membrane sodium channels. The data presented herein show that voltage sensitive sodium channels as defined by specific activation by alkaloid neurotoxins are qualitively different in insect CNS synaptosomes than those described in vertebrate brain synaptosornes, nerve membrane preparations and cultured neuronal cells. REFERENCES Breer H. and Knipper M. (1985) Synaptosomes and neuronal membranes from insects. In Neurochemical Techniques in Insect Research (Edited by Breer H. and Miller T. A.), PP. 1255155. Sorineer. Berlin. Catterall W. A. (i977) Activatibn if the action potential Na+-ionophore by neurotoxins: an allosteric model. J. biol. Chem. 252, 8669-8676. Catterall W. A. (1986) Molecular properties of voltagesensitive sodium channels. A. Rev. Biochem. 55, 953-985. Catterall W. A. and Nirenberg M. (1973) Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma and muscle cells. Proc. nam. Acad. Sri. U.S.A. 17, 3759-3763. Couraud F., Martin-Moutot N.. KoulakotT A. and Berwald-Netter Y. (1986) Neurotoxin-sensitive sodium channels in neurons developing in cico and in vitro. J. Neurosci. 6, 192-198. Dwivedy A. K. (1985) Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroach, Periplaneta americana. Pesric. Sci. 16, 615-626. Dwivedy A. K. (1986) Sodium channels of the cockroach central nervous system synaptosomes. In Insect Neurochemistry and Neurophysiology (Edited by Borkovec A. B. and Gelman D. B.), pp. 393-396. The Humana Press, _. _ New Jersey
354
A. K. DWIVEDY
Dwivedy A. K. (1987) Neurotoxin-stimulated sodium transport in an insect central nervous system synaptosomes. Biochem. Sot. Trans. 15, 145-146. Ghiasuddin S. M. and Soderlund D. M. (1984) Mouse brain synaptosomal sodium channels: activation by aconitine. batrachotoxin, and veratridin, and inhibition by tetrodotoxin. Comp. Biochem. Physiol. 7X, 267-271.. Grischenko I. I.. Naumov A. P. and Zubov A. N. (1983) Gating and selectivity of aconitine-modified sodium‘chan: nels in neuroblastoma cells, Neuroscience 9, 549-554. Harris R. A. and Bruno P. (1985) Effects of ethanol and other intoxicant anesthetics on voltage-dependent sodium channels of brain synaptosomes. J. Pharmacol. exp. Ther. 232, 4OlL406. Khodorov B. 1. (1985) Batrachotoxin as a tool to study voltage-sensitive sodium channels of excitable membranes Prog. Biophys. mol. Biol. 45, 57-148. Krueger B. C. and Blaustein M. P. (1980) Sodium channels in presynaptic nerve terminals: regulation by neurotoxins. J. gen. Physiol. 16, 287-313. Lazdunski M. and Renaud J. F. (1982) The action of cardiotoxins on cardiac plasma membrane. A. Rev. Physiol. 44, 463-470. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-215. Narahashi T. (1982) Pharmacology of ionic channels in excitable membranes. Adc. Pharmac. Ther. 11, 3-18. Narahashi T.. Moore J. R. and Shapiro B. 1. (1969) Condylactis’toxin: interaction with ne&e membrane ionic conductance. Science 163, 680-68 I. Naumov A. P. (1983) Modification of sodium channel with scorpion toxins and alkaloids. In Toxins as Tools in Neurochemistry (Edited by Hucho F. and Ovachinnikov Yu.), pp. 13-23. Walter de Gruyter and Co., Berlin. Pelhate M. and Sattelle D. B. (1982) Pharmacological properties of insect axons. A Review. J. Insect Physiol. 28, 889-903.
Pichon Y. (1976) Pharmacological properties of the ionic channels in insect axons. In-Peri$aneta in Experimental Biology (Edited by Spencer Davies P.)., DD. . . 297-312. Pergamon Press, dxford. Pichon Y. and Treherne J. E. (1974) The effects of sodium transport inhibitors and cooling on membrane potentials in cockroach central nervous connectives. J. exp. Biol. 61, 203-218. Rashatwar S. and Matsumura F. (1985) Reduced calcium sensitivity of the sodium channel and the Na+/Ca+ exchange system in the Kdr-type, DDT and pyrethroid resistant German cockroach, Blattella Germanica. Camp. Biochem. Physiol. UC, 97-103. Romey G., Abita J. P., Schweitz H., Wunderer G. and Lazdunski M. (1976) Sea anemone toxin: a tool to study molecular mechanisms of nerve conduction and excitation-secretion coupling. Proc. nain. Acad. Sci. U.S.A. 73, 4055.-4059. Romey G. and Lazdunski (1982) Lipid soluble toxins thought to be specific for Na+ channel blocks CaZ+ channels in neuronal cells. Nafure 297, 79-80. Rosenberg R. L., Tomiko S. A. and Agnew W. S. (1984) Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus. Proc. natn. Acad. Sci. CI.S.A. 81, 123991243. Schofield P. K. and Treherne J. E. (1975) Sodium transport and lithium movements across the insect blood-brain barrier. Nature 255, 7233725. Talvenheimo J. A., Tamkun M. M. and Catterall W. A. (1982) Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain. J. biol. Chem. 257, 11868-l 1871. Tamkun M. M. and Catterall W. A. (1981) Ion flux studies of voltage-sensitive sodium channels in synaptic nerveending particles. Mol. Pharmacol. 19, 78-86. Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics. B&hem. J. 80, 324-332.
Eiochem.
Physiol.
Vol.
91C.
No.
2. pp.
349-354,
0306~4492/88
1988
ALKALOID TRANSPORT
$3.00
+ 0.00
Pergamon Pressplc
Printed in Great Britain
NEUROTOXINS-DEPENDENT SODIUM IN INSECT SYNAPTIC NERVE-ENDING PARTICLES
A. K. DWIVEDY A.F.R.C. Unit of Insect Neurophysiology and Pharmacology, Dept. of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK (Telephone (0223) 336600) (Received
21 August 1987)
Abstract-l. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplunefa americana) was investigated. of Na+ uptake; 2. Batrachotoxin (K,,, , 0.2 FM) was full agonist as for most effective activator of “Na+ uptake veratridine (K, 5, 2.5 PM) and aconitine (K,,,, 7.6 p M) produced a maximal stimulation that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I, s. I I nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins-stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect svnantosomes than those oreviouslv described for vertebrate brain synaptosomes, cultured neuronal cell, _ nerve membrane vesicles and neuroblastoma cells. 1
variations in the properties of sodium channels. In insects and crustaceans, the access of neurotoxins has been available only to the external surface of axonal preparations. The responses of some neurotoxins are ineffective in cephalopod axons (Romey et al., 1976), though are effective in cockroach and crustacean nerves (Narahashi et al., 1969; Pelhate and Sattelle, 1982). The present investigation describes appropriate conditions for measuring sodium channeldependent **Na+ influx in the synaptosomal fraction isolated from the central nervous system (CNS) of the cockroach, Periplaneta americana. The responses of alkaloid toxins, batrachotoxin (BTX), veratridine (VTN), and aconitine (ACN) which activate specifically to the influx of sodium in insect presynaptic membranes are described. Some of the results have been presented in part elsewhere (Dwivedy, 1986; Dwivedy, 1987).
INTRODUCTION sodium channels play a key role in generation of action potential in many excitable membranes. The detection and modification in the properties of sodium channels have been described with the use of a number of classes of neurotoxins from plant and animal origin (Lazdunski and Renaud, 1982; Catterell, 1986). Neurotoxins are highly specific in their affinity with the receptors of sodium channels and have been defined predominantly as sodium channel blockers (tetrodotoxin, saxitoxin), sodium channel inactivation inhibitors (sea anemone toxin, scorpion toxin) and sodium channel modifiers (batrachotoxin, veratridine, aconitine). The last group of toxins are lipid soluble and have been refereed as alkaloid neurotoxins (Catterall, 1977; Romey and Lazdunski, 1982). Alkaloid neurotoxins, unlike other neurotoxins, affect voltage-sensitive sodium channels simultaneously at the activation, inactivation, ion selectivity and susceptibility of many drugs and toxins (Narahashi, 1982; Naumov, 1983; Khodorov, 1985). Alkaloid neurotoxin-stimulated sodium uptake has been described among a variety of neuronal membrane preparations by measuring **Na+ influx in synaptosomes from rat brain (Krueger and Blaustein, 1980; Tamkun and Catterall, 1981) mouse brain (Ghiasuddin and Soderlund, 1984), cells from mouse neuroblastoma (Catterall, 1977), cultured neurons from rat brain (Couraud et al., 1986), liposomes preparation from rat brain (Talvenheimo et al., 1982) and electroplax of EIectrophorus electricus (Rosenberg et al., 1984). These preparations differ in their response to alkaloid toxins, and further comparative studies are of considerable interest to the knowledge of species-dependent Voltage-sensitive
MATERIALS AND METHODS Chemicals 22NaCI (carrier free, 450mCi/mg) was purchased from Amersham International, UK. Tetrodotoxin (TTX), aconitine (ACN), veratridine (VTD), ouabain, choline chloride, Albumin (fatty acid free bovine serum albumin, BSA), N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES), ethylenediaminetetraacetic acid dipotassium salt (EDTA), ethyleneglycol-bis(-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) and [tris (hydroxymethyl) aminomethane] (Tris) were purchased from S&a Chemicals, UK. Pot&sium cyanide (I&N) was obtained from Fisons Scientific UK. Batrachotoxin (BTX) was a generous gift of Dr John W. Daly, NIH, Bethesda; MD, U.S.A. Deionized and purified reagent grade water was used to prepare all reagent solutions. Preparation of synaptosomes Synaptosomes and abdominal
c 1988 Crown copyright. 349
were prepared from dissected head, thorax ganglion of adult male cockroach, P.
350
A. K.
americana, according to the method as previously described (Dwivedy, 1985, 1987). The insect ganglion were pooled, homogenized in ice-coid 0.25 M sucrose, containing 0.1 M Tris~ch~oride, and 1.0 mM EDTA (pH 7.4). The homogenate was centrifuged (4‘C) at 8OOg for 15 min. The pellet was homogenized twice with the buffered sucrose solution and recentrifuged. The supernatant were combined and centrifuged at 10,SOOg for 20 min. The resultant pellet (P,) was suspended in sucrose buffer (0.1 ml), homogenized gently with 0.25 M sucrose (0.5 ml) containing 13% Ficoll and suspension (0.5 ml) transferred to Beckman uhramicrocentrifuge tubes of 0.7 ml capacity. Buffered sucrose (0.1 ml of 0.2 M sucrose, containing 1.0 mM K-EDTA, pH 7.4) was added gently on top of sucrose- Ficoll mixture, and allowed to equilibrate at 4’C before it was centrifuged (4°C) at 9500g for 60min. The synaptosomal fraction was carefully collected from the interface with micropipette and resuspended in 0.25 M sucrose buffer, and suspension was centrifuged (15,OOOg for 20 min) to obtain the synaptosomes in pellet form. Influx of *2Na+ in the synaptosomes was measured by a slight modification of procedure as described (Dwivedy, 1987). The synaptosomal preparation was suspended in a sodium free low calcium buffer (5 mM glucose, 3 mM MgCl,, 8 mM KCl, 0.5 mM CaCl,, 0.5 mM EGTA, 2 mM ouabain, 2 mM KCN, 20mM Hepes-Tris. pH 7.4). Solutions of appropriate concentrations of toxins and **Na (2mM. 1-2 uCi/ml) both were made in incubation buffer (20 mM choiine ‘chloride, mg/ml BSA, added to suspension buffer used for suspending synaptosomal fraction, pH 7.4). Aliquots of synaptosomal suspension (10 ill, IO-20 pg) were incubated initially with appropriate concentration of toxins for i min in a total volume of 190~1 incubation buffer. The uptake of zzNa+ in synaptosomes was measured after addition of 10~1 of 2zNa+ into the media having a total volume of 200~1 which was equilibrated in a water bath maintained at 21°C. influx measurements were terminated at desired length of time intervals by aspirating off the radioactive assay medium, transferring it onto buffer soaked filter paper (Whatman GF/C) followed by immediate vacuum filtration (Dwivedy, 1987). Since a significant loss of *‘Na+ occurred with the use of various wash medium at different time intervals (Fig. l), each incubation tube and filters were washed twice as rapidly as possible (less than 30 see) with about 1.0 ml of ice cold wash solution (5 mM glucose, 0.8 mM MgSO,, 1.8mM CaCl,, 20 mM choline chloride, 20 mM Hepes-Tris, pH 7.4). Filters were air dried, dissolved in 20% methyl cellosolve, and counted in ACS (Amersham) liquid scintillation (75% counting efficiency). Specific *jNaT uptake was calculated by substracting the non-specitic zzNa+ uptake obtained in presence of TTX and VTD (sodium-channel activator) from that obtained with VTD alone. Measurement of internal r~olumeqf .synuptosomes The intracellular volume of synaptosomes was measured with tritiated PHI water and ~‘4Clinuiin as internal and external volume markers respectively according to the method described by Breer and Knipper (1985). About 80-100 pg of synaptosomal protein -was &tbated for 10 min at 4°C in a reaction medium (0.5 ml) with a mixture of radiolabelled tracer (10 FCi jH,O + “C-carboxyi inulin, 1-2 p Ci/ml). After incubation, the mixture was diluted with 0.5 ml of reaction buffer. It was incubated for 5 min at room temperature, and centrifuged at 10,800g for 5 min in an Eppendorf centrifuge. From each tube, supernatant and residual pellet were separated, both dissolved separately in 0.5 ml of 1% SDS solution and washed with 1.0 ml of buffer before appropriate amount was counted, and internal volume caiculated. The interval volume of synaptosomes was expressed relative to the protein. The protein content of the
DWIVEDY
fraction were measured by the method of Lowry et al. (1951) using crystalline BSA as the standard. RESIJLTS
Eflect of ouabain on f2Na + injh.~
Inhibition of Nat, K+-ATPase by ouabain and synthesis of ATP by KCN both affect the influx of sodium when synaptosomes are exposed to 2mM external sodium concentration. In absence of ouabain (Fig. 2A), there was a relatively low “Na+ influx in synaptosomes (O.lLO.2 nmol/sec/mg of protein). However, with graded increase in the molar concentration of ouabain, the inward movement of **Nat increased and apparently equilibrated at approximately external concentration of 2 x lO-‘M ouabain. The time course of 22Na+ influx in presence of 2 x 10-l M ouabain (Fig. 2B) indicated a linear curve reached between 30-60sec, in contrast to neurobfastoma cells (30-6Omin) when incubated with 5 mM ouabain (Catterail and Nirenberg, 1973). The cell volume measurement suggested synaptosomal intracellular volume about 2.5 pll/mg of protein. Since 22Na+ uptake in presence of 2 x IO-‘M ouabain approached to 0.36 nrno~/se~~rng of synaptosomal protein, the final intracellular concentration of 22Na+ was 0.15 mM to the extracellular concentration of 2.0 mM sodium. The difference between the extents of uptake of ‘*Na+ in presence and absence of ouabain (Fig. 2B) would suggest that synaptosomes maintain an internal Na’ concentration of 0.1-0.2 mM against an externai concentration of 2mM sodium used in the present study. Assay conditions
Effect of sodium concentrations on initial rates of VTD-stimulated (SOpM) specific Z2Na+ uptake is shown in Fig. 3. VTD-stimulated specific 12Na+ uptake was linear only when the sodium concen-
100 aa .!G
9
3
::4 g
rn
80 tb . GO-
.fj
40 -
I a
20 -
O.Ok 0.0
1.0
2.0
3.0
5.0
Time (min)
Fig. 1. Effect of wash medium on the efflux of ‘“Na+. Synaptosomes (15-20 ~8 protein) were incubated in 2 mM 22NaCi (I pCi/ml) at 2 1’ with 50 ,UM veratridine for 30 sec. transferred to filter paper (Whatman GF/C), followed by vacuum filtration, and then rinsed at given length of time intervals in a wash medium consisting of 5 mM glucose. 0.8 mM MgSO,, 1.8 mM CaCI,, 20mM Hepes-Tris, pH 7.4, with either 20 mM NaCl and 8 mM KC1 (O), or 20 mM choline chloride and 8 mM KC1 (0). or 20 mM chofine chloride (O), or 160 (A). The . . mM choline^ chloride . contmuous lme IS a least-square tit to the data
351
Sodium transport in insect synaptosomes (A)
(B)
L4---+-0.0
1o-4
1O-2
10
I Ouabain I ( M )
L00 .
.
.
5.0
.
Time (akin)
Fig. 2. (A) Effect of ouabain concentration on ‘%a+ uptake in the cockroach CNS synaptosomes (O.Ol-0.02mg of protein). Uptake of “Na+ (2mM, IL2~Ci/ml) was measured for 30sec at 2l’C in presence of indicated concentrations of ouabain. (B) Time effect on uptake of j*Na+ in the synaptosomes in absence (V) or presence of 2.0 mM ouabain (0). Each point represents the average of duplicate experiments.
was increased up to 2mM in incubation media. This suggests that insect synaptic membranes have saturable binding sites of sodium channels. Moreover, a higher concentration than 2 mM of sodium in incubation may result increased sodium permeability which could induce depolarization and reduce in electrical driving force for ‘*Na+-influx. The range of protein content for each incubation were about 0.01 S-O.02 mg of synaptosomal protein. Effect of wash medium on the efficacy of “Na+ efflux suggest that a significant loss of sodium would have occurred if wash medium contained 160 mM choline chloride (Fig. 1). A wash medium containing 8 mM KC1 either with 20 mM NaCl or with 20 mM choline chloride effected the rapid loss of sodium. A wash medium consisting of 20mM choline chloride was used maximally for 30 set in each experiment. tration
Time course of sodium channel activation by VTD Sodium channel specific uptake of *‘Na+ in the synaptosomes were determined by subtracting nonspecific “Na+ uptake from VTD-stimulated *?Na+ influx. The initial rate of **Na.+influx in presence of VTD (50 p M) was greatly enhanced than the rate of
non-specific influx of Na+ (in absence of VTD), and deviation in the linearity of specific *‘Nat influx was observed between 30 set to 1 min (Fig. 4). At the same period of incubation, the VTD-stimulated influx of ‘*Nat was inhibited by 1OpM TTX. Since the experimental values for incubation times less than 30 set were highly variable, and there was no appreciable increase in specific influx of ‘*Na-’ after 30 set of incubation, standard incubation time of 30 set was opted for in all subsequent experiments. Sodium channel activation ofBTX, VTD and ACN The alkaloid toxin BTX, VTD and ACN all produced concentration dependent sodium uptake in the synaptosomes (Fig. 5). Values for the toxinconcentration giving a half maximal activation (K,,,) and maximal activation obtained for each toxin are given in Table 1. BTX was a potent stimulator for synaptosomal sodium uptake which produced **Na+ influx increase to 13 and 38 fold higher than that produced for VTD and ACN respectively over nonspecific uptake at maximal activation. VTD and
0.9, 0.8
-
Cl
0
2.0
6.0
4.0
INa+J
tM
x
10.0
IO-~)
Fig. 3. Effect of sodium concentration for veratridinedependent (50pM) *%a+ uptake in the cockroach CNS synaptosomes (0.01-0.02 mg of protein). Uptake of **Na+ was measured for 30 set as described in methods. Symbols represent means + SEM, n = 3 experiments.
I
0.3
k
1.0
I
1
1.5
2.0
I
3.0
Time (min)
Fig. 4. Time courses of 22Na+ uptake in the cockroach CNS synaptosomes (0.01-0.02 mg protein). Non-specific uptake of 22Na+ was measured at 21°C as described under methods in the absence of toxins (a), uptake in presence of 50pM veratridine (e), and in the presence of 50pM veratridine plus IOpM TTX (A). The data points represent the means + SEM (n = 3).
A. K.
352
DWIVEDY DISCUSSION
5
6
I
6
[Activators1
The present result demonstrated that neurotoxins BTX, VTD. and ACN activate the flux of sodium through the voltage dependent sodium channels of insect synaptosomes and are inconsistent with the voltage clamp studies on activation of sodium channels of cockroach giant axons by some alkaloids of plant origins (Pichon, 1976; Pelhate and Sattelle, 1982). Alkaloid neurotoxins induced a steady state of Na+-permeability reactions of the action potential in the insect synaptosomes and the concentration at which TTX inhibited the generation of action potential in insect axon was apparently of same the range that inhibited the “Nat uptake in the cockroach CNS synaptosomes (Fig. 6). However, the movement of “Nat across nerve membrane depends on both the external and internal ion concentrations and the variable factors such as Na ’ K +-ATPase and Na+/Ca + exchange transporter which may influence the sodium channel gating kinetics. Electrophysiological studies have shown that ouahain inhibits the movement of sodium across insect axons though it was insensitive to sodium influx in perineurium and underlying glial cells (Pichon and Treherne, 1974; Schofield and Treherne, 1975). In the present study, a low intracellular steady state of Na +-concentration within synaptic membrane was maintained by the inhibition in the activity of Na + , K +-ATPase. For the measurement of sodium channel-dependent ‘2Na’ influx, insect synaptosomes were routinely incubated in ouabain, KCN and EGTA; and a suitable washing procedure for incubated synaptosomes was followed. A low concentration of sodium for incubation was determined so that membrane potential is unaffected by the increased sodium permeability caused by the neurotoxin treatment. Appropriate experimental conditions for incubation of synaptosomes resulted in initial rates of non-specific “Nat flux considerably lower than the values obtained using otherwise similar conditions in German cockroach nerve membrane preparation (Rashatwar and Matsumura. 1985).
3
4
(-10 log MI
Fig. 5. Concentration--effect curves for alkaloids toxin dependent 22Na+ uptake (difference between the uptake in presence and absence of toxins) in the cockroach CNS synaptosomes (O.Ol--0.02mg of protein). Uptake was measured at indicated concentrations. Symbols represent the means 2 SEM. n = 4, BTX (a). VTD (I). ACN (A).
ACN both were less effective giving 71% and 43% of the activation observed for BTX at maximally effective concentrations (Table I). 2zNa + uptake by TTX
Inhibition of sped@
The VTD-stimulated (50 p M) uptake of *‘Na + was inhibited by TTX in a concentration dependent manner (Fig. 6). The apparent IC,, for TTX was about 11 nM, and a higher concentration above IO !itM of TTX completely blocked VTD-stimulated “Na + influx.
Table
I.
Comparison
vesicles.
cultured
of the activakw
mouse
neuronal
of sodium
cella.
and
channels
mouse
of vertebrate
neuroblastoma
brain
cells
bv
synaptosomes.
BTX.
VTD
brain
and
membrane
ACN.
ND.
not
Actrvators BTX
VTD
CNS synaptosomes
Insect
(present Mouse
brain
synaptosomes:
brain
synaptosomes$
Rat
brain
synaptosomes~
Cultured 112.
neuroblastomaq mouse i3-dav
neuronal
fetal
*Concentration activation $Data
from
activation of Ghiasuddin
cell**
giving
a
equation available
(100)t
2.5
0 5 (71)
75
$,
SCtiVBllOll
c
(I 7 (43)
IlData
of Krueger
‘IData
of Catterall of Courdud
0.4
8.0 (100)
34.5
5.9 (73)
19.6
3.7 (46)
0.5
0.9 (100)
13.0
0.5 (55)
14.0
0.2 (22)
3.0
0.6 (50)
0.5
1.2(100)
0.7
0.9 (100)
29.0
0.1 (II)
0.2
132.7 (100)
10.4
67.3 (51)
ND 3.6
0.02 (2) ND
half-maximal according
(nmol/sec/mg and
of protein).
Soderlund
and Blaustein (1977).
to
activation. Wilkinson
A (1961)
basic
computer
in evalualing
programme the
values
for
was
designed for
&,,
and
maximai
data.
gData of Tamkun and Catterall
**Data
0.7
activation
brain1
(PM)
Michaelis-Menten tMaximal
0 2
Maximal
K/bi
study)
Rat
Mouse
actwation
..-__K,, <*
______-..
ACN
MaxImal
Maximal
lnmtal maximal
(1981).
maximal
(1980).
maximal
maximal
et al. (1986).
(1984).
activation maximal
rate m absence activation
activation activation
of activator
(nmol/min!mg
(nmol:min/mg
of protein).
(nmol!min/mg
of protein).
(nmol/min/culture).
activation
was 0.1 nmol:sec;mg of protein).
(nmol/min:lO*
cells).
of protein.
353
Sodium transport in insect synaptosomes
-100
-75
- 50
-25
p
B. @ E 4 6 ,x 3
-0
[TTX]
(610losM)
Fig. 6. The effect of different concentrations of tetrodotoxin on the veratridine induced (50 PM) **Na+ uptake in the synaptosomes (O.OlLO.02 mg of protein). The indicated concentrations of tetrodotoxins were added, and **Na+ uptake was measured as described. The data are presented as the means k SEM, n = 4.
The present study CNS synaptosomes
on
sodium
transport
in insect
demonstrates a concentration dependence of alkaloid neurotoxins for the activation of voltage sensitive sodium channels-ionophore. The potency of BTX-stimulated sodium transport was greater (about ten times that of VTD) and similar to the magnitude of that observed for the synaptosomes from the brain of rat and mouse (Table 1). The VTD-stimulated sodium uptake in the synaptosomes from rat cortex, rat cerebellum and mouse brain was found to be 2.6, 1.2 and 1.9 nmol/mg of protein/set respectively and the concentration giving a half maximal activation for both BTX and VTDstimulated influx of **Na+ was found to be dependent on temperature-giving a lower value at the higher temperature (Harris and Bruno, 1984). The other factors likely to contribute to the differences in the rate of specific *‘Na+-flux may include different sodium concentration used and differences in the composition of loading media. Neurotoxins affect the kinetics of sodium channel acivation during membrane depolarization as suggested by a shift in the maximum sodium conductance in nerve membranes (Grischenko et al., 1983; Khodorov, 1985). BTX interacts preferentially with open channels and their modification is fairly stable, while that by VTD is not stable. and ACN pronounced inactivation of ACN-modified sodium channels provides the reason for the fact that ACN produced much smaller increases in “Nat-flux in synaptosomes than did VTD and BTX. The activation of sodium channels of cockroach CNS synaptosomes by BTX, VTD and ACN is compared to that of vertebrate brain synaptosomes, cultured neuronal cells, and neuroblastoma cells in Table I. It indicates the differences in the potency of neurotoxins, BTX being a full agonist, VTD and ACN both partial agonists in insect CNS synaptosomes similar to the observations in other nerve membrane preparations. However, the neurotoxins produced characteristic different responses in sodium channels of insect nerve membrane preparations for both to the values of K, 5 and the extent of maximal activatton. In particular,
the partial agonists VTD and ACN had a greater intrinsic activity in activating sodium channel to 71% and 41% of BTX-maximal activator as compared with SO-%% and 2-22% in rat brain synaptosomes and 5 I % and 2% in neuroblastoma cells. The study suggests that insect synaptosomes, a subcellular membrane preparation of CNS of Peripluneta americana, retain functional properties that are identical with those of intact nerve cells, and provide an ideal subcellular preparation for studying the functional and structural properties of nerve membrane sodium channels. The data presented herein show that voltage sensitive sodium channels as defined by specific activation by alkaloid neurotoxins are qualitively different in insect CNS synaptosomes than those described in vertebrate brain synaptosornes, nerve membrane preparations and cultured neuronal cells. REFERENCES Breer H. and Knipper M. (1985) Synaptosomes and neuronal membranes from insects. In Neurochemical Techniques in Insect Research (Edited by Breer H. and Miller T. A.), PP. 1255155. Sorineer. Berlin. Catterall W. A. (i977) Activatibn if the action potential Na+-ionophore by neurotoxins: an allosteric model. J. biol. Chem. 252, 8669-8676. Catterall W. A. (1986) Molecular properties of voltagesensitive sodium channels. A. Rev. Biochem. 55, 953-985. Catterall W. A. and Nirenberg M. (1973) Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma and muscle cells. Proc. nam. Acad. Sri. U.S.A. 17, 3759-3763. Couraud F., Martin-Moutot N.. KoulakotT A. and Berwald-Netter Y. (1986) Neurotoxin-sensitive sodium channels in neurons developing in cico and in vitro. J. Neurosci. 6, 192-198. Dwivedy A. K. (1985) Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroach, Periplaneta americana. Pesric. Sci. 16, 615-626. Dwivedy A. K. (1986) Sodium channels of the cockroach central nervous system synaptosomes. In Insect Neurochemistry and Neurophysiology (Edited by Borkovec A. B. and Gelman D. B.), pp. 393-396. The Humana Press, _. _ New Jersey
354
A. K. DWIVEDY
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Pichon Y. (1976) Pharmacological properties of the ionic channels in insect axons. In-Peri$aneta in Experimental Biology (Edited by Spencer Davies P.)., DD. . . 297-312. Pergamon Press, dxford. Pichon Y. and Treherne J. E. (1974) The effects of sodium transport inhibitors and cooling on membrane potentials in cockroach central nervous connectives. J. exp. Biol. 61, 203-218. Rashatwar S. and Matsumura F. (1985) Reduced calcium sensitivity of the sodium channel and the Na+/Ca+ exchange system in the Kdr-type, DDT and pyrethroid resistant German cockroach, Blattella Germanica. Camp. Biochem. Physiol. UC, 97-103. Romey G., Abita J. P., Schweitz H., Wunderer G. and Lazdunski M. (1976) Sea anemone toxin: a tool to study molecular mechanisms of nerve conduction and excitation-secretion coupling. Proc. nain. Acad. Sci. U.S.A. 73, 4055.-4059. Romey G. and Lazdunski (1982) Lipid soluble toxins thought to be specific for Na+ channel blocks CaZ+ channels in neuronal cells. Nafure 297, 79-80. Rosenberg R. L., Tomiko S. A. and Agnew W. S. (1984) Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus. Proc. natn. Acad. Sci. CI.S.A. 81, 123991243. Schofield P. K. and Treherne J. E. (1975) Sodium transport and lithium movements across the insect blood-brain barrier. Nature 255, 7233725. Talvenheimo J. A., Tamkun M. M. and Catterall W. A. (1982) Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain. J. biol. Chem. 257, 11868-l 1871. Tamkun M. M. and Catterall W. A. (1981) Ion flux studies of voltage-sensitive sodium channels in synaptic nerveending particles. Mol. Pharmacol. 19, 78-86. Wilkinson G. N. (1961) Statistical estimations in enzyme kinetics. B&hem. J. 80, 324-332.