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Enhancement of norepinephrine release from rat brain synaptosomes by alpha cyano pyrethroids
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
28, 127- 139 (1987)
Enhancement of Norepinephrine Release from Rat Brain Synaptosomes by Alpha Cyano Pyrethroids MATTHEW Department
of Entomology.
W. BROOKSAND University
J. MARSHALLCLARK~
of Massachusetts,
Amherst,
Massachusetts
01003
Received September 23. 1986; accepted January 12, 1987 Using a continuous perfusion system, synaptosomes prepared from rat brain readily took up L-[7,8-3H]norepinephrine and released it in a calcium-dependent, potassium-stimulated manner. Of the insecticides tested, only pyrethroids which contained an alpha cyano grouping as part of their phenoxybenzyl alcohol moiety (i.e., deltamethrin. cypermethrin, and fenvalerate) enhanced calcium-dependent, potassium-stimulated release of [3H]norepinephrine. Non-cyano-pyrethroids (Le., allethrin, des-cyano deltamethrin, des-cyano cypermethrin, and des-cyano fenvalerate) were greatly reduced in their ability to enhance [3H]norepinephrine release. None of the pyrethroids tested had any effect on [3H]norepinephrine uptake at concentrations at or below 10es M. None of the pyrethroids tested at concentrations up to 10msM had any effect on [3H]norepinephrine release from nondepolarized synaptosomes. The ED,, dose of deltamethrin which resulted in half-maximal-enhanced [3H]norepinephrine release (i.e.. 2.9 x lO-9 M) correlated well with the ED,, dose of deltamethrin which resulted in half-maximal-enhanced 45Ca uptake (i.e., 2.4 x lo-!’ M. Parathion and the noninsecticidal DDT analog, DDE, had no effect on [3H]norepinephrine uptake or release. DDT was found to be the most potent inhibitor of [3H]norepinephrine uptake: tc 1987 Academic
Press. Inc.
INTRODUCTION
Two distinct symptoms of poisoning by pyrethroid insecticides have been described in rats. Animals poisoned with non-cyano-pyrethroids (i.e., type I) elicit a tremor or T syndrome whereas animals poisoned with alpha cyano pyrethroids (i.e., type II) elicit choreoathetotic writhing and profuse salivation (i.e., CS syndrome) (1). The CS syndrome results in electroencephalogram changes indicative of CNS involvement, particularly within the extrapyramidal motor system (2). Also, a direct correlation between the levels of deltamethrin in the brain and the onset of CS symptomology has been determined (3). These and many other results indicate that the primary site of action of pyrethroids in mammals is within the CNS (4-7). Because the convulsive state is so evident in the symptomology of pyrethroid
r To whom all correspondence should be addressed, at Department of Entomology, University of Massachusetts, Fernald Hall, Amherst, MA 01003.
toxicity, involvement of central monoaminergic systems with various convulsive states becomes most interesting. The depletion of catecholamines within the CNS causes a significant reduction in threshold levels that are necessary to induce both electroconvulsive shock (8, 9) and pentylenetetrazole-induced (10) seizures. Reductions of these thresholds are apparently more highly correlated to reduction of norepinephrine than to dopamine (11). Recently, a variety of drugs that interfere either in synthesis or in release of norepinephrine were found to potentiate pyrethroid toxicity significantly (6). Additionally, induction of a type II syndrome by deltamethrin caused a substantial enhancement of plasma norepinephrine concentration in the blood of treated rats (12). A similar enhancement was not apparent in cismethrin-treated rats. Thus, an enhanced release of norepinephrine due to the action of alpha cyano pyrethroids could lead to an overall depletion of brain stores of this neurotransmitter, producing a convulsive state typical of this type of pyrethroid poisoning. 127 0048-3575187 $3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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The present study details those experimental conditions that resulted in an enhanced release of norepinephrine from rat brain synaptosomes pretreated with various insecticides in support of this contention. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats, 56 to 60 days old, were obtained from Charles River Breeding Laboratories Inc., Wilmington, Massachusetts. Chemicals. The following chemicals were gifts as indicated: deltamethrin ((S)-acyano-3-phenoxybenyl-cis-( lR,3R)-2,2-dimethyl-3-(2,2-dibromovinyl)cyclopropanecarboxylate) from Dr. J. Martel, Roussel Uclaf, Romainville, France; des-cyano deltamethrin from Dr. D. M. Soderlund, New York State Agricultural Experimental Station, Geneva, New York; cypermethrin ((+)-IX-cyano-3-phenoxybenzyl-( +)-cis, trans-3-(2.2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) from Shell Bioscience Laboratory, Sittingbourne, Kent, United Kingdom; permethrin (3-phenoxybenzyl( +), cis trans-3-(2,2-dichlorovinyl)2,2 - dimethyl - cyclopropanecarboxylate) from FMC Corp., Middleport, New York. Fenvalerate ((S)-cx-cyano-3-phenoxybenzyl-2-(4-chlorophenyl)-isovalerate) and des-cyano fenvalerate were from Dr. M. E. Schroeder, Shell Development Co., Modesto, California. S-Bioallethrin ((2,2-dimethyl)-3-(2-methyl1-propenyl) cyclopropanecarboxylic acid-2-methyl-4-0x0-3-(2propenyl)-2-cyclopenten1-yl-ester) was from Dr. I. Yamamoto, Tokyo University of Agriculture, Tokyo, Japan; DDT (l,l, ltrichloro-2,2-bis(p-chlorophenyl)ethane, DDE (1,l -dichloro-2,2-bis(p-chlorophenyl)ethene, and parathion (O,O-diethylO-p-nitrophenyl phosphorothioate) were purchased from Chem Services, West Chester, Pennsylvania. L-[7,8-3H]norepinephrine (r3H]NE,* 30-40 Ci/mmole) and 45CaC12 (17 mCi/mg) were obtained from 2 Abbreviations nephrine; NSM,
used: [3H]NE, normal superfusion
L-[7,VH]norepimedia; DM,
po-
CLARK
Amersham Radiochemicals, Arlington Heights, Illinois, and New England Nuclear, Boston, Massachusetts, respectively. D600 (5[3,4-dimethoxyphenylethyl)methylamino]- 2 -isopropyl- 2 -(3,4,5trimethoxyphenyl)valeronitrile hydrochloride) and D595 (5-[(3,4-dimethoxyphenylethyl)methylaminol-2-(3,4-dichlorophenyl)-2-isopropylvaleronitrile hydrochloride) were gifts of Professor Kretzschmar and Professor Oberdorf, Knoll AG, West Germany, respectively. All other organic and inorganic chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri. Preparation of synaptosomes. Synaptosomes were prepared from whole brains by the method of Whittaker et al. (13) as modified by Hajos (14). Male rats were sacrificed by cervical dislocation and whole brains were removed. Brain matter with a wet weight of approximately 3-4 g was disrupted using a Teflon-glass homogenizer in 20.0 ml 0.32 M sucrose and then centrifuged. The P, fraction (crude mitochondrial fraction) was resuspended in 5.0 ml 0.32 M sucrose, layered onto a discontinuous sucrose density gradient of 0.32, 0.8, and 1.2 M sucrose, and centrifuged at 50,OOOg for I hr. The material at the 0.8- 1.2 M interface was collected by aspiration. This was returned to a more physiological normotonic environment by addition of small volumes of ice-cold normal superfusion media (NSM, Table 1) over a 30-min period to a final volume equal to four times the collected synaptosomal volume (15). The equilibrated synaptosomes were then pelleted at 15,OOOg for 10 min and resuspended in 2.0 ml ice-cold NSM. The concentration of protein was set typically between 8 and 11 mg/ml (16). tassium-depolarizing media: OCaNSM, zero-calcium normal superfusion media: OCaDM, zero-calcium potassium-depolarizing media; LCNSM, low-calcium normal superfusion media: LCDM, low-calcium depolarizing media; D600, S-[(3.4-dimethoxyphenylethyl)methylamino]-2-isopropyl-2-(3.4,5-trimethoxyphenyl)valeronitrile hydrochloride; D595 5-[(3,4-dimethyloxyphenylethyl)methylamino]-2-(3.4-dichlorophenyl~L-isopropyl valeronitrile hydrochloride.
NOREPINEPHRINE TABLE
RELEASE
BY ALPHA
I
Representative Buffer Compositions of External Solutions0 (mmoles/Liter) Solutic& NSM DM OCaNSM OCaDM LCNSM LCDM
N&l 128 77 128 77 128 77
KC1
D-Ghcose
5 56 5 56 5 56
16 16 16 16 17 17
CaCl,
M&I,
I 1 0.01 0.01
I I -
Ba’&
-
* In addition, all solutions contained 12 ~,44 nialamide and 20 mM Hepes. All solutions were adjusted to pH 7.35 with Tris base. * NSM, normal superfusion media; DM. depolarization media; OCa-. zero-calcium buffers; LC-. low-calcium buffers.
Uptake and release of [3H]NE. The procedure for uptake and release of 13HlNE was identical to that developed by Levi and Raiteri (17) as modified by West and Fillenz (18). Synaptosomal aliquots (0.1 ml) were incubated at 37°C with 0.139 kiI4 [3H]NE (approx. 111, 446 cpm per aliquot) for 15 min with constant shaking. Uptake was terminated by addition of 1.0 ml icecold NSM. Background radiation was determined from blanks which consisted of identical incubations carried out at 0°C. All solutions were filtered with suction onto 0.65-km cellulose acetate filters (Millipore Co., Bedford, MA) and washed with 10 ml of ice-cold NSM to remove any unbound [3H]NE. Uptake of [3H]NE was determined by extracting filters overnight in I M perchloric acid (1.0 ml) and measuring the extracted (acid-solubilized aliquot) and nonextracted (filter) by liquid scintillation spectrometry. Specific uptake was determined by subtracting the total cpm value of the 0°C incubation from that which occurred at 37°C. Release of [3H]NE was measured using a continuous perfusion device that consisted of eight IO-ml polypropylene syringes fixed in a container which was maintained at 37 ? 2°C. Synaptosomal aliquots on cellulose acetate filters were housed in 25-mm Swinnex propylene filter holders (Millipore Co.) which were attached to the bottom of each syringe barrel. Tygon tubing carried perfused liquid from filters to scintillation vials via peristaltic pumps. There existed a
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3-ml dead volume in the tubing. Hence at a flow rate of 0.5 ml/min, it would take fluid approximately 6 min to appear in the scintillation vial. Twenty-four OS-ml fractions were collected over a 24-min period from each barrel. At fraction 6 (6 min), selected barrels were depolarized by addition of 2.0 ml KC depolarizing media (DM, Table I). Depolarization was terminated after 3 min by diluting the remaining solution IO-fold by addition of NSM. The radioactivity (cpm t3H]NE) in each fraction was determined by liquid scintillation spectrometry. Calculation of [3HjNE released. Release of [3H]NE was expressed either as a fractional rate constant or as a summation of fractional rate constant differences. The fractional rate constant is the amount of [3H]NE released in each I-min fraction (i.e., 0.5 ml) as a percentage of the radioactivity remaining in synaptosomes during the preceding minute (19-22). After determining fractional rate constants for all barrels, a fractional average of all fractional rate constants from similarly treated barrels was calculated. The fractional average determined for nondepolarized barrels was then subtracted from the fractional average determined for depolarized barrels to give a fractional average difference. The fractional average differences for fractions 12 through 20 were summed and these summations of fractional average differences (e.g., summations of [3H]NE released) were used to compare the effects of various insecticides and inhibitors. Addition
of inhibitors
and insecticides.
In experiments which used water-insoluble inhibitors or insecticides, compounds were solubilized in 95% ethanol. Their amounts were adjusted to give a final assay concentration when 1 .O ~1 was added to a 0. l-ml synaptosomal aliquot. Synaptosomes were preequilibrated for 10 min on ice prior to the addition of [3H]NE. Ethanol was included in all control tubes and accounted for not more than 1% of the total volume. Tetrodotoxin was solubilized in 6.0 x 10m3 M sodium citrate (pH = 4.8). Sodium ci-
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trate solution was added to control tubes (assay concentration was 6 x lop5 M>. Tetraethylammonium was solubilized in distilled water. In experiments with insecticides, incubation tubes were coated with Carbowax PEG 20000. Insecticides were added to carbowax-treated tubes and incubated under standard conditions. Solutions were transferred to noncoated tubes prior to loading with [3H]NE. This transfer was necessary in that carbowax (polyethylene glycol) readily absorbs amines such as norepinephrine. Calcium-45 uptake studies. Synaptosomes were preincubated with either deltamethrin or 1.0 ~1 of 95% ethanol prior to addition of unlabeled norepinephrine (0.139 kiV NE). The incubation with unlabeled NE was performed as described for [3H]NE. Uptake of unlabeled NE was terminated after 15 min by addition of 10.0 ml ice-cold low-calcium normal superfusion media (LCNSM, Table 1). Aliquots were filtered with suction onto 0.65~km Millipore filters and perfused as described with an additional 3.0 ml of LCNSM. Selected barrels received 2.0 ml of low-calcium depolarization media (LCDM) containing 0.1 &i 45CaCl,/ml (approx. 250,000 cpm). The remaining barrels received LCNSM which also contained 0.1 uCi 45CaCl,/ml. After a 3-min 45Ca perfusion period, all filters were washed with 15.0 ml ice-cold LCNSM and the 45Ca present on filters was determined by liquid scintillation spectrometry. Statistics. T tests and least-squares regression analyses were performed on the cyber CDC mainframe computer located at the University of Massachusetts using the Statistical Package for the Social Sciences (SPSS). RESULTS
Characterization of synaptosomal uptake of r3aNE. Uptake of [3H]NE is carried out by two distinct mechanisms: a high affinity-active transport mechanism known as uptake 1 (24) which is ouabain sensitive
CLARK
and dependent on the presence of external Na+, and a low-affinity transport system known as uptake 2 (25) which is ouabain insensitive and less dependent on external Na+. Table 2 shows that both of these uptake mechanisms are functional in the present preparation. Uptake of [3H]NE in the presence of ouabain (10e3 M> was inhibited 60 ? 4% when compared with control values. When Na+ was replaced by choline on an equimolar basis, uptake was inhibited 64 + 3% when compared with control values. Therefore, approximately 60% of [3H]NE taken up by synaptosomes was by the high-affinity uptake 1 mechanism. The remaining 40% of uptake was ouabain insensitive and independent of external Na+ concentration which is in agreement with the uptake 2 mechanism. The average uptake to [3H]NE for these experiments was 3.82 2 0.59 pmoles/mg protein/ 15 min. The remaining results summarized in Table 2 reveal that modification of ion fluxes has little effect on [3H]NE uptake. Addition of the K+ channel blocker, tetraethylammonium, the Cl- channel blocker, picrotoxinin, or the Ca2+ channel blockers, D600 and D595, had no effect on synaptosomal uptake of [3H]NE. Effect of insecticides on synaptosomal uptake of [3H]NE. Pyrethroids had little or no significant (P < 0.05) effect on uptake of [3H]NE by synaptosomes at concentrations of lop5 M or less (Table 3). Of the pyrethroids tested at 1O-5 M, only fenvalerate resulted in a significant inhibition of [3H]NE uptake but reduction was only 11% of the control value. The organochlorine, DDT, was the most inhibitory insecticide tested, resulting in a 21% inhibition of control [3H]NE uptake at 10e6 M. Neither DDE nor parathion had any significant effect on synaptosomal uptake of [3H]NE at concentrations up to 10e4 M. Characterization of synaptosomal release of [3HjNE. Functionally intact synaptosomes can be artificially depolarized by increasing external concentrations of K+
NOREPINEPHRINE
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TABLE 2 Effect
of Biochemical
Inhibitors
on [‘H]NE
Uptake
into Rat Brain
Synaptosomes
[3H]NE uptake Concentrationa
Inhibitor
Ouabain Choline Tetrodotoxin Tetraethylammonium Picrotoxinin D600 D595
Control
(pmoles [3H]NE + SEMlmg protein/l5 min) 4.45 t 0.05 1.78 2 4.45 t 0.05 1.62 2 3.32 ” 0.23 3.71 k 3.32 r 0.23 3.53 k 3.43 r 0.30 4.25 f 4.45 t 0.05 5.00 k 3.32 -c 0.23 3.52 2
10m3 M 128 x 10m3M lo-‘M
5 x 1O-3 M 2 x 1O-5 M IO-*
Treated
M
1O-5 M
O.l6*J’ 0.11” 0.18 0.20 0.38 0.46 0.01
N = 3’ N=3 N=2 N=3 N=2 N=3 N=2
u Control synaptosomes were treated with 1.O ~195% ethanol. Treated synaptosomes received the concentration of inhibitor as listed in 1.O pl 95% ethanol. b Means followed by an asterisk are significantly (P < 0.05) different from control means (one-tailed t test). c N denotes number of experiments. Each experiment contained four replicates.
ions which in the presence of Ca*+ leads to release of vesicle-bound neurotransmitters (18, 26,27). When viable synaptosomes are continuously perfused and pulsed by depolarized K+ in the presence of Ca*+. they release neurotransmitter in a measurable and reproducible pattern (2, 8). The data in Fig. 1 demonstrate that rat brain synaptosomes utilized in this work are viable and reproduce a similar pattern of neurotrans-
mitter (e.g., [3H]NE) release under the present experimental conditions. Only synaptosomes in the presence of Ca2+ showed an enhanced rate of t3H]NE release when K+ depolarized (Fig. IA). This is illustrated by the increase in fractional rate constants for fractions (min) 12 through 20, inclusively. Synaptosomes which were perfused in media containing Mg2+ instead of Ca2+ (open circles) or which never re-
TABLE Effect
of Insecticides (pmol
on [3HjNE Uptake [3HjNE ? SEMimg
3 into Rat Brain Synaptosomes protein/l5 min)
[3H]NE uptake Insecticide
Control”
1O-6 M
1O-5 M
1O-4 M
Deltamethrin Fenvalerate Cypermethrin des-Cyano Deltamethrin des-Cyano Fenvalerate Allethrin Permethrin DDT DDE Parathion
3.44 k 0.19 3.24 k 0.47 3.23 k 0.13
3.42 r 0.15 3.04 * 0.14 3.48 2 0.13
3.66 -e 0.37 2.87 k 0.06* 3.47 f 0.22
2.53 i 0.19*.b 2.13 2 0.26* 3.30 + 0.12
3.43 2 0.17 3.43 3.43 4.02 3.45 3.23 3.23
k k -c f 2 k
0.17 0.07 0.16 0.09 0.03 0.03
4.55 k 0.07 3.19 4.71 2.71 3.05 3.36
2 2 t 2 k
0.14 0.07 0.09* 0.02 0.16
4.73 3.49 4.64 3.00 3.11 3.83
2 + L + k i
0.06 0.24 0.15 0.1s* 0.31 0.09
N = 4’ N=3 N=3 N=3
2.66 3.94 2.28 3.19 3.06
f 2 2 i 2
0.30* 0.27 0.13* 0.09 0.13
N=3 N=5 N=3 N=3 N=3 N=3
a Control synaptosomes were treated with 1.0 ~195% ethanol. b Means followed by an asterisk are significantly different from control means (P < 0.05, one-tailed t test). c N represents number of experiments. Each experiment consisted of four replicates.
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CLARK 12lo-
;;
A
+ D’ta/ I. Control 8
b
-depolarized
non-depolarized
IL’
6
2
4
6
B
10 12
14 16 1E 20 22
24
FractiondMid FIG. 1. Effect of calcium on the release of [3HjNE. Synaptosmal aliquots M’ere loaded Jc*ith 0.139 (IM 13HlNE in media containing 1 mM Ca2+ and subsequently tc,ashed and perfused wjith media containing either 1 mM Ca2+ (NSM. closed circles) or 1 mM Mg2+ (OCaNSM, open circles). (A) All synuptosomnl aliquots were depolarized ufter 6 min (jiiaction 6, as indicated by arrow) by addition qf media containing 56 mM K+ and either Ca2+ or Mg2+ (DM or OCaDM, respectitaely) for 3 min. (B) The K+ level remained at 5 mM throughout the perfksion (i.e.. nondepolarized). EfJux MWS expressed as a ,fructional rate constant. The fractional rate constants are determined by dividing cpm released during that fraction by the cpm present in the synaptosomal aliquot ut the beginning of the fraction. The results represent an coverage of four experiments titith each experiment consisting of four replicates.
ceived K+-depolarizing media (Fig. lB), regardless of the presence of Ca2+, did not change their rates of [3H]NE release. Under these experimental conditions, the average calcium-dependent, potassiumstimulated release of [3H]NE was calculated to be 70 + 12 fmoles [3H]NE/mg protein/3 min. This value represents approximately 1.8% of total [3H]NE uptake. Effect
of deltamethrin
on [3H]NE
re-
lease. Figure 2 shows the fractional rate constants calculated for [3H]NE release from synaptosomes treated with lop5 M of either deltamethrin (Dlta, open circles) or ethanol (Control, closed circles). Addition of K+-depolarization media to delta-
FractiondMin) FIG. 2. Effect of deltamethrin on [3H]NE efflux. Synaptosomal aliquots were incubated with either lo-= M deltamethrin (Dlta. open circles) or I.0 ~195% ethanol (control, closed circles) prior to loading with 0.139 PM [3H]NE. Aliquots were subsequently naashed and perfked with NSM. (A) Synuptosomes were depolarized by addition of DM for 3 min (us indicated by arrow). (B) Synuptosomes were treated tixith NSM for the entire assay. Results are expressed as ,fractionul rate constants (see Fig. I ). The results represent an average of four experiments nsith each experiment consisting offour replicutes.
methrin-treated synaptosomes produced a large initial spike of [3H]NE release compared with ethanol-treated synaptosomes (Fig. 2A). The initial enhanced spike following K+ depolarization suggests that deltamethrin facilitates Ca2+ entry into the cytosol (22). Furthermore, depolarization of deltamethrin-treated synaptosomes produced a prolonged tailing effect of [3H]NE release which continued through fraction 20. Enhanced release of [3H]NE was not evident in synaptosomes which did not receive K+ depolarization regardless of the presence of deltamethrin or ethanol (Fig. 2B). Thus, increased [3H]NE release due to the addition of deltamethrin was evident only during depolarization and in the combined presence of Ca2+. As illustrated in Fig. 3, the effect of deltamethrin is dose dependent. Synapto-
NOREPINEPHRINE
RELEASE
BY ALPHA
to-i0 Deltamethrln
CYAN0
10-g Concentration
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PYRETHROIDS
10-7
10-s CM)
FIG. 3. Effect of various concentrations of deltamethrin on release of [‘HINE. Synaptosomal fractions were incubated toith either deltamethrin (crossed bars) or I .O $957~ ethanol (open bars) prior to loading with 0.139 FM [3HjNE. Aliquots were subsequently washed and perfused tijith NSM. After 6 min (fraction 6), selected aliquots were depolarized with DM for 3 min. Results are expressed as a summation of [‘HjNE released (see Materials and Methods section). Both nondepolarized and depolarized aliquots received similar treatments (i.e., either all received deltamethrin or all received 95% ethanol). The results represent an average of two experiments k standard error measurement (SEM). Each experiment consisted offour replicates.
somes treated with deltamethrin at lo-” and lo-lo M gave [3H]NE release values (i.e., summation of [3H]NE released) upon depolarization that were not significantly different (P < 0.05) from control values (13.6 L 0.1% for lo-lo A4 compared with 13.7 i 2.3% for controls, and 12.9 ? 2.0 for lo-” M compared with 14.7 + 1.0% for controls). Enhanced [3H]NE release apparent at lop9 M deltamethrin was significantly different from control values (P < 0.05) (treated synaptosomes released 13.2 L 0.3% compared with 9.7 ~fr.0.2% for controls). Maximum stimulation was achieved at lo-’ M deltamethrin (treated synaptosomes released 27.8 + 3.7% compared with 12.6 ? 0.5% for controls) and this response became saturated at higher concentrations of deltamethrin. The average enhanced release of [3H]NE in the presence of lo-’ M deltamethrin for these experiments was calculated to be 115 2 7.1 fmoles [3H]NE/mg protein/3 min which
represents approximately 3.01% of the total [3H]NE load. Effect of deltamethrin on 45Ca uptake. Since norepinephrine release has been demonstrated to be highly dependent on Ca*+ influx, an attempt was made to directly measure the effect of deltamethrin on Ca*+ uptake. As illustrated in Fig. 4, deltamethrin enhanced synaptosomal uptake of 45Ca during K+ depolarization in a dose-dependent manner (crossed bar) over control values (solid bar). Significant enhancement (P < 0.05) was noted with deltamethrin concentrations greater than lo-lo M. Potassium-stimulated 45Ca uptake reached a maximum enhanced level at a deltamethrin concentration of lop7 M (550 ? 50 fmoles 45Ca/mg protein/3 min) with saturation noted at lop5 M. The control value of K+-stimulated 45Ca uptake for these experiments was calculated to be 250 i 55 fmoles 45Ca/mg protein/3 min. To correlate the effect of deltamethrin on
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Deltamethrin
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Concentration
(Ml
4. Effect of deltamethrin on %a uptake. Synaptosomal aiiquots tizere incubated JO min M’itJl either deltamethrin or I.0 ~1 95% ethanol. Synaptosomes were loaded with 0.139 )LM unlabeled NE, washed, and perfused with NSM. After 6 min (fraction 6). selected fractions were depolarized by the addition of 2.0 ml of LCDM containing 0.1 PCi of 4sCa per milliliter (solid plus crossed bars). The remaining aliquots received 2.0 ml of LCNSM containing 0.1 @i of “Y?a per milliliter (open bars). After 3 min, all synaptosomal aliquots were washed with 15.0 ml LCNSM, and the radioactivity remaining on the filters teas determined by liquid scintillation spectrometty. Solid burs represent the amount of ‘Yu tuken up bv depolarized synaptosomes which Msere preincubated with 95% ethanol. Crossed bars represent any additional amount of ‘Vu uptake which w’as in excess of the control 1~11~ due to the presence of deltamethrin. The mean values are abarrages & SEM of two experiments, euch consisting of four replicates. FIG.
[3H]NE release and 45Ca uptake, enhanced values of [3H]NE release (closed circles) and 45Ca uptake (open circles) due to increasing the concentration of deltamethrin from lo-lo to IO-’ M were subjected to a regression analysis and co-plotted (Fig. 5). The effective dose of deltamethrin which resulted in a 50% maximal response (i.e., ED,,) for both these phenomena was estimated from these regressed lines. In the case of enhanced [3H]NE release (solid line), the ED,, for deltamethrin was calculated to be 2.9 x 1O-9 M compared with 2.4 x 10m9 M for enhanced 45Ca uptake (dashed line). The apparent stoichiometry between 45Ca uptake and [3H]NE release estimated from these lines is approximately 5.8 Ca2+ to 1 NE. Comparison of the effect of various in-
secticides on [3HJNE release. As judged by the summation value of [3H]NE released for fractions 12 to 20, deltamethrin was the most effective compound in enhancing L3H]NE release. Deltamethrin enhanced 13H]NE release 114% compared with an untreated control value (Fig. 6A). Other alpha cyano pyrethroids were also effective in enhancing release. Cypermethrin resulted in a 64% enhancement while fenvalerate enhanced [3H]NE release 40% above the control value. The relative ability of deltamethrin, cypermethrin, and fenvalerate to enhance [3H]NE release is calculated as 1:0.56:0.35, respectively. This ratio correlates well with the relative toxicity ratio calculated from the acute oral LD,, values for rat (i.e., deltamethrin = 135 mg/kg, cypermethrin = 251 mg/kg, and
NOREPINEPHRINE
RELEASE
Deltamethrin
BY ALPHA
Concentration
CYAN0
PYRETHROIDS
135
(M)
FIG. 5. Least-squures regression analysis of deltamethrin-stimulated [‘H]NE release (@) and 4SCa uptake (0). Using the same experimental protocol as detailed in Figs. 3 and 4, respectively, six values of enhanced [3HJNE release and 4sCa uptake at various concentrations of deltamethrin (from lO&‘O to lo-’ M) were regressed andplotted. The regression analysis for enhanced [3H]NE release (-) dae to the addition of deltamethrin gave an rz value of 0.82, and 0.98 f or 45Ca uptake (--). Each data point represents the mean of 01’0 experiments which consisted offoar replicates each.
fenvalerate = 451 mg/kg) (30) which results in a ratio of 1:0.54:0.30. Pyrethroids that lacked an alpha cyano grouping were greatly reduced in their ability to enhance [3H]NE release (Fig. 6B). Addition of bioallethrin resulted in a 4.7% enhancement and des-cyano cypermethrin in a 1.2% enhancement but neither was significantly different (P < 0.05) from control values. Des-cyan0 deltamethrin and des-cyano fenvalerate were also without an effect. Table 4 summarizes the effect of DDT, DDE, and parathion on [3H]NE release during K” depolarization. At 10es M, DDT produced a highly significant (P < 0.05) effect resulting in a 92% inhibition of [3H]NE release compared with controls. At the concentration, however, DDT has been determined to significantly inhibit synaptosomal [3H]NE uptake (see Table 3). Neither parathion nor DDE at concentrations of
lops M had any significant (P < 0.05) effect on synaptosomal efflux of [3H]NE. DISCUSSION
Enhanced release of [3H]NE from rat brain synaptosomes by alpha cyano pyrethroids is evident only in the combined presence of external Ca*+ and membrane depolarization. This indicates that insecticides like deltamethrin act as use-dependent compounds (31-34). Because release of [3H]NE is highly dependent on Ca*+ entering the cytosol of the synaptosome, it can be assumed that deltamethrin may act on entities which transport Ca*+, such as the voltage-sensitive sodium and calcium channels (29, 35). In a similar study, [3H]NE was released from rat brain cortex slices by electrical stimulation. This release was shown to be absolutely dependent on extracellular calcium and was blocked not only by tetrodotoxin but also by the cal-
BROOKS
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CLARK
FIG. 6. Effect of various pyrethroid insecticides on release of [3HjNE. Synaptosomal aliqaots were incubated with either a 10m5 M concentration ofpyrethroid (crossed bars) or 1.0 ~195% ethanol (open bars) prior to loading with 0.139 @I [3H]NE. Aliquots were subsequently washed and perfused with NSM. After 6 min (fraction 6), selected aliquots were depolarized with DM. (A) The effects of alpha cyano pyrethroids (type II). (B) The effects of pyrethroids lacking such a moiety (type I). Results are expressed as a summation of [‘HjNE released -t SEM (see Materials and Methods section). Summations expressed are an average sf ttijo experiments, each consisting offour replicates.
cium channel antagonist, D600, and by manganese (22). Ghiasuddin and Soderlund (32) have determined a Kd for [3H]descyano deltamethrin binding to mouse brain synaptosomes of 4 x lop8 M. Because del-
Effeecr of Nonpyrethroid
Insecticides K+-Depolarized
[3H]NE Insecticide”
DDT DDE Parathion
TABLE 4 on Ca 2f-Dependent Synaptosomes
a The concentration of all insecticides was * Control synaptosomes were treated with c Relative insecticide-induced NE release of control value. d Values represent means of two experiments asterisk are significantly (P < 0.05) different
Treated release
[3HJNE
Release
? SEM)d 1.2 ? 2.4* 14.1 + 1.3 12.5 2 1.3
10m5 M. 1 .O ~1 95% ethanol. is the r3H]NE released
from
Relative insecticide-induced [3H]NE release (percentage of control released)c
release
Control* (summation [3H]NE 15.4 & 0.8 15.2 * 1.8 14.5 * 2.1
tamethrin is 10 times more toxic, it can be assumed that it is also 10 times more potent and hence has 10 times greater affinity for its receptor. If this assumption is true, deltamethrin’s apparent Kd should be in the
7.8 92.7 86.2
from
treated
synaptosomes
with four replicates for each insecticide. from control means (one-tailed t test).
Means
as a percentage followed
by an
NOREPINEPHRINE
RELEASE
BY ALPHA
order of 4 x 10e9 M. This compares well with the ED,, of deltamethrin of approximately 3 x 1O-9 M which was calculated for both [3H]NE release and 45Ca uptake in this study. Electroencephalographic studies have shown that alpha cyano pyrethroids exert a highly selective action at the caudate nucleus in the corpus striatum of the brain (36). The increased caudate blood flow caused by intraperitoneal administration of deltamethrin preceded the development of electroencephalogram spike discharges and the development of motor symptoms typical of type II pyrethroids. Cismethrin. a type I pyrethroid, did not produce enhanced blood flow in the caudate nucleus. Interestingly, brain arterioles and capillaries are innervated by adrenergic nerve fibers indicative of a central adrenergic control of cerebral blood flow (37). These results correlate well with those presented initially by Aldridge et al. (38), who showed that acetylcholine was significantly depleted from rat brain after oral administration of deltamethrin. Cismethrin was greatly reduced in its ability to deplete this rictu-otransmitter while DDT resulted in no depletion whatsoever. Recently, it has been shown that only the toxic s-acid, s-alcohol isomer of fenvalerate invoked an enhanced neurotransmitter release in rabbit brain striatal slices (39). A similar enhanced neurotransmitter release was not apparent in rabbit brain hippocampal slices. In related studies that examined the effects of several pyrethroids and DDT on insect neuromuscular preparations, it was reported that alpha cyano pyrethroids were very potent in increasing the rate of miniature excitatory postsynaptic potentials. DDT and non-alpha-cyan0 pyrethroids were much reduced in their ability to increase the rate of potential firing (40). Recently, deltamethrin has been shown to cause almost complete depletion of synaptic vesicles from presynaptic motor nerve terminals of the house fly. This indi-
CYAN0
PYRETHROIDS
137
cates a presynaptic action of deltamethrin (41). Interestingly, mitochondria of treated terminals were reported to be swollen and have vacuolated interiors. It is well established that mitochondria function as a major calcium regulator within presynaptic nerve terminals (15). One possible explanation which links the effect of deltamethrin on [3H]NE release to pyrethroid symptomology is that by causing the synaptosome to rapidly lose [3H]NE, the nerve terminal is depleted of NE stores. Reduction of NE in the brain results in loss of blood vessel constriction and ultimately in increased blood flow. Depletion of brain NE stores has been shown to be highly correlated to the convulsive state (9, 1I), a condition evident in deltamethrin poisoning. Finally, compounds which have been shown to deplete monoaminergic neurotransmitters like norepinephrine significantly potentiate the toxicity of pyrethroids (6). Thus, there appears to be a correlation of enhanced [3H]NE release, depletion of brain stores of NE, increased cerebral blood flow, and the convulsive state, all of which are caused by the action of deltamethrin. These results are in accordance with the theory that voltagesensitive ionic channels in nerve cells are the primary target of pyrethroids (3). As shown by Vijverberg and de Weille (42). the most pronounced effect of alpha cyano pyrethroids on nerve cells under voltageclamp conditions is the prolongation of the time constants calculated for the decay of sodium tail currents. Most notable is the fact that all alpha cyano pyrethroids result in greatly enhanced time constants reflecting extremely slow kinetics of the tail currents. The addition of an alpha cyano grouping to lR-trans-permethrin (which yields lR-trans-cu-S-cypermethrin) caused the time constant to increase from 7.3 to 1 I14 msec, respectively. Prolonged opening of voltage-sensitive ionic channels, both sodium and calcium, could enhance calcium influx and result in enhanced neu-
138
BROOKS AND CLARK
rotransmitter release, and ultimately in the depletion of synaptic vesicles and neurotransmitters in presynaptic nerve terminals. Indeed, our data on enhancement of norepinephrine release by alpha cyano pyrethroids (i.e., deltamethrin 114%, cypermethrin 64%, and fenvalerate 40% over control release) when converted to a relative ratio (e.g., 1:0.56:0.35) compares well with Vijverberg and de Weille’s data on time constant enhancement by alpha cyano pyrethroids (i.e., 1772: 1114:603 msec of 1:0.63:0.34, respectively). It is not expected that non-alpha-cyan0 pyrethroids would be as effective in creating a prolonged depolarization as are the alpha cyano pyrethroids.
9.
10.
11.
12.
13. ACKNOWLEDGMENTS This work was supported by research Grant RR07048-19, NIH-BRSG, and by the Massachusetts Agricultural Experimental Station, UMASS, Amherst. Massachusetts. REFERENCES 1. R. D. Verschoyle and W. N. Aldridge. Structureactivity relationships of some pyrethroids in rats, Arch. Toxicol. 45, 325 (1980). 2. D. E. Ray, An EEG investigation of decamethrin induced choreoathetosis in the rat, Exp. Brain Res. 38, 221 (1980). 3. J. Rickard and M. Brodie, Correlation of blood and brain levels of the neurotoxic pyrethroid deltamethrin with the onset of symptoms in rats, Pestic. Biochem. Physiol. 23, 143 (1985). 4. D. E. Ray and J. E. Cremer. The action of decamethrin (a synthetic pyrethroid) on the rat, Pestic. Biochem. Physiol. 10, 333 (1979). 5. J. E. Cremer, V. J. Cunningham, D. E. Ray, and G. S. Sarna. Regional changes in brain glucose utilization in rats given a pyrethroid insecticide, Brain Res. 194, 278 (1980). 6. C. G. Staatz, A. S. Bloom, and J. J. Lech. A pharmacologic study of pyrethroid neurotoxicity in mice, Pestic. Biochem. Physiol. 17, 287 (1982). 7. C. G. Staatz-Benson and M. J. Hosko. Interaction of pyrethroids and mammalian spinal neurons, Pestic. Biochem. Physiol. 25, 19 (1986). 8. A. D. Rudzik and G. A. Johnson, Effect of amphetamine and amphetamine analogs on convulsive thresholds, in “International Symposium on Amphetamines and Related Com-
pounds” (E. Costa and S. Garattini, Eds.), p. 715, Raven Press, New York, 1970. M. Kilian and H. H. Frey, Central monoamines and convulsive thresholds in mice and rats, Neuropharmacology 12, 681 (1973). I. Maj and J. Vetulani, Some pharmacological properties of N.N-disubstituted dithiocarbamates and their effect on the brain catecholamine levels, Eur. J. Pharmacol. 9, 183 (1970). A. Quattrone, V. Crunelli, and R. Samanin, Seizure susceptibility and anticonvulsant activity of carbamazepine, diphenyldantoin and phenobarbitol in rats with selective depletions of brain monoamines, Neuropharmacology 17, 643 (1978). J. E. Cremer and M. P. Seville, Comparative effects of two pyrethroids, deltamethrin and cismethrin, on plasma catecholamines and on blood glucose and lactate. Toxicol. Appl. Pharmacol. 66, 124 (1982). V. P. Whittaker, 1. A. Michaelson, and R. J. Kirkland, The separation of synaptic vesicles from nerve ending particles (synaptosomes). Biothem.
J. 90, 293 (1964).
14. F. Hajos. An improved method for the preparation of synaptosomal fractions in high purity, Brain Res. 93, 485 (1975). 15. M. P. Blaustein, R. W. Ratzlaff, and N. K. Kendrick. The regulation of intracellular calcium and presynaptic nerve terminals, Ann. N. Y. Acad. Sci. 307, 195 (1978). 16. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. M. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 17. G. Levi and M. Raiteri, Synaptosomal transport processes, Int. Ret,. Neurobiol. 19, 51 (1976). 18. D. P. West and M. Fillenz, Storage and release of noradrenaline in hypothalamic synaptosomes, J. Neurochem. 3X6), 1323 (1980). 19. F. Orrego, J. Jankelevich, C. Ceruti, and E. Ferrera, Differential effects of electrical stimulation on release of 3H-noradrenaline and i4Caminoisobutyrate from brain slices, Nature (London) 251, 55 (1974). 20. F. Orrego and R. Miranda, Electrically induced release of 3H-GABA from neocortical thin slices, J. Neurochem. 26, 1033 (1976). 21. G. Levi, V. Gallo, and M. Raiteri, A reevaluation of veratradine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings, Neurochem. Res. X3), 281 (1980). 22. F. Orrego and S. Sanchez-Armass, Electrically induced release of 3H-NE from rat brain cortex slices: A kinetic analysis of the dependence of
NOREPINEPHRINE
RELEASE
BY ALPHA
extracellular
calcium, Pharmacol. Res. 13(10), 949 (1981). D. W. Helmuth, S. M. Ghiasuddin, and D. M. Soderlund, Poly(ethylene glycol) pretreatment reduces pyrethroid adsorption to glass surfaces, j. Agric. Food Chem. 31, 1127 (1983). L. L. Iverson, Role of transmitter uptake mechanisms in synaptic transmission, &it. J. Pharmacol. 41, 571 (1971). .I. S. Gillespie. Uptake of noradrenaline by smooth muscle, Brit. Med. BulL. 29, 136 (1973). M. P. Blaustein, Effects of potassium, veratradine, and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro, /. Physiol. 247, 617 (1975). G. Maura, A. Pittaluga. A. Ricchetti. and M. Raiteri, Noradrenaline uptake inhibitors do not reduce the presynaptic action of clonidine on 3Hnoradrenaline release in superfused synaptosomes, Naunyn-Schmied Arch. Pharmacol. 327, 86 (1984). M. Raiteri, F. Angelini, and G. Levi, A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Phnrmacol. 25, 411 (1974). R. R. Llinas, Calcium in synaptic transmitter, Sci. Amer. 287(4), 56 (1982). C. R. Worthing, “The Pesticide Manual,” 7th ed.. British Crop Protection Council, U.K., 1983. A. E. Lund and T. Narahashi, Kinetics of sodium channel modifications as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs, Pestic. Biochem. Common.
23.
24. 25. 26.
27.
28.
29. 30. 31.
Physiol.
34.
35. 36. 37.
38.
39.
40.
41.
20, 203 (1983).
32. S. M. Ghiasuddin and D. M. Soderlund, Pyrethroid insecticides: Potent, stereospecific enhancers of mouse brain sodium channel activation, Pestic. Biochem. Physiol. 24, 200 (1985). 33. G. S. F. Ruigt. Pyrethroids, in “Physiology, Biochemistry and Pharmacology” (G. Kerkut and
42.
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L. Gilbert, Eds.), Vol. 12, pp. 184-251, Pergamon, New York, 1985. T. Narahashi, Nerve membrane ionic channels as the primary target of pyrethroids, Neurotoxicology 6(2), 3 (1985). L. J. Mullins, Calcium entry upon depolarization of nerve, J. Physiol. (Paris) 77, 1139 (1981). D. E. Ray, Changes in brain blood flow associated with deltamethrin-induced choreoathetosis in the rat, Exp. Brain Res. 45, 269 (1982). R. Nieuwenhuys, J. Voogd, and C. Van Huijzen, “Human Central Nervous System,” pp. 218-219, Springer-Verlag. 1981. W. N. Aldridge, B. Clothier, P. Forshaw. M. K. Johnson. V. H. Parker, R. J. Price, D. N. Skilleter. R. D. Verschoyle. and C. Stevens, The effect of DDT and the pyrethroids cismethrin and dehamethrin on the acetylcholine and cyclic nucleotide content of rat brain Biochem. Pharmacol. 27, 1703 (1978). .I. T. Eells and M. L. Dubocovich, Release of neurotransmitters from rat brain slices by fenvalerate, in “6th Int. Cong. Pestic. Chem. (IUPAC),” Abstr. No. 3A-16, Ottawa, Canada, 1986. V. L. Salgado, S. N. Irving, and T. A. Miller, The importance of nerve terminal depolarization in pyrethroid poisoning of insects, Pestic. Biothem. Physiol. 20, 169 (1983). L. P. Schouest, Jr., B. L. Salgado, and T. A. Miller. Synaptic vesicles are depleted from motor nerve terminals of deltamethrin-treated house fly larvae, Muscu domestica. Pestic. Biochem. Physiol. 25, 381 (1986). H. P. M. Vijverberg and J. R. de Weille, The interaction of pyrethroids with voltage-dependent Na channels, Neurotoxicology 6, 23 (1985).
BIOCHEMISTRY
AND
PHYSIOLOGY
28, 127- 139 (1987)
Enhancement of Norepinephrine Release from Rat Brain Synaptosomes by Alpha Cyano Pyrethroids MATTHEW Department
of Entomology.
W. BROOKSAND University
J. MARSHALLCLARK~
of Massachusetts,
Amherst,
Massachusetts
01003
Received September 23. 1986; accepted January 12, 1987 Using a continuous perfusion system, synaptosomes prepared from rat brain readily took up L-[7,8-3H]norepinephrine and released it in a calcium-dependent, potassium-stimulated manner. Of the insecticides tested, only pyrethroids which contained an alpha cyano grouping as part of their phenoxybenzyl alcohol moiety (i.e., deltamethrin. cypermethrin, and fenvalerate) enhanced calcium-dependent, potassium-stimulated release of [3H]norepinephrine. Non-cyano-pyrethroids (Le., allethrin, des-cyano deltamethrin, des-cyano cypermethrin, and des-cyano fenvalerate) were greatly reduced in their ability to enhance [3H]norepinephrine release. None of the pyrethroids tested had any effect on [3H]norepinephrine uptake at concentrations at or below 10es M. None of the pyrethroids tested at concentrations up to 10msM had any effect on [3H]norepinephrine release from nondepolarized synaptosomes. The ED,, dose of deltamethrin which resulted in half-maximal-enhanced [3H]norepinephrine release (i.e.. 2.9 x lO-9 M) correlated well with the ED,, dose of deltamethrin which resulted in half-maximal-enhanced 45Ca uptake (i.e., 2.4 x lo-!’ M. Parathion and the noninsecticidal DDT analog, DDE, had no effect on [3H]norepinephrine uptake or release. DDT was found to be the most potent inhibitor of [3H]norepinephrine uptake: tc 1987 Academic
Press. Inc.
INTRODUCTION
Two distinct symptoms of poisoning by pyrethroid insecticides have been described in rats. Animals poisoned with non-cyano-pyrethroids (i.e., type I) elicit a tremor or T syndrome whereas animals poisoned with alpha cyano pyrethroids (i.e., type II) elicit choreoathetotic writhing and profuse salivation (i.e., CS syndrome) (1). The CS syndrome results in electroencephalogram changes indicative of CNS involvement, particularly within the extrapyramidal motor system (2). Also, a direct correlation between the levels of deltamethrin in the brain and the onset of CS symptomology has been determined (3). These and many other results indicate that the primary site of action of pyrethroids in mammals is within the CNS (4-7). Because the convulsive state is so evident in the symptomology of pyrethroid
r To whom all correspondence should be addressed, at Department of Entomology, University of Massachusetts, Fernald Hall, Amherst, MA 01003.
toxicity, involvement of central monoaminergic systems with various convulsive states becomes most interesting. The depletion of catecholamines within the CNS causes a significant reduction in threshold levels that are necessary to induce both electroconvulsive shock (8, 9) and pentylenetetrazole-induced (10) seizures. Reductions of these thresholds are apparently more highly correlated to reduction of norepinephrine than to dopamine (11). Recently, a variety of drugs that interfere either in synthesis or in release of norepinephrine were found to potentiate pyrethroid toxicity significantly (6). Additionally, induction of a type II syndrome by deltamethrin caused a substantial enhancement of plasma norepinephrine concentration in the blood of treated rats (12). A similar enhancement was not apparent in cismethrin-treated rats. Thus, an enhanced release of norepinephrine due to the action of alpha cyano pyrethroids could lead to an overall depletion of brain stores of this neurotransmitter, producing a convulsive state typical of this type of pyrethroid poisoning. 127 0048-3575187 $3.00 Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
128
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The present study details those experimental conditions that resulted in an enhanced release of norepinephrine from rat brain synaptosomes pretreated with various insecticides in support of this contention. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats, 56 to 60 days old, were obtained from Charles River Breeding Laboratories Inc., Wilmington, Massachusetts. Chemicals. The following chemicals were gifts as indicated: deltamethrin ((S)-acyano-3-phenoxybenyl-cis-( lR,3R)-2,2-dimethyl-3-(2,2-dibromovinyl)cyclopropanecarboxylate) from Dr. J. Martel, Roussel Uclaf, Romainville, France; des-cyano deltamethrin from Dr. D. M. Soderlund, New York State Agricultural Experimental Station, Geneva, New York; cypermethrin ((+)-IX-cyano-3-phenoxybenzyl-( +)-cis, trans-3-(2.2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) from Shell Bioscience Laboratory, Sittingbourne, Kent, United Kingdom; permethrin (3-phenoxybenzyl( +), cis trans-3-(2,2-dichlorovinyl)2,2 - dimethyl - cyclopropanecarboxylate) from FMC Corp., Middleport, New York. Fenvalerate ((S)-cx-cyano-3-phenoxybenzyl-2-(4-chlorophenyl)-isovalerate) and des-cyano fenvalerate were from Dr. M. E. Schroeder, Shell Development Co., Modesto, California. S-Bioallethrin ((2,2-dimethyl)-3-(2-methyl1-propenyl) cyclopropanecarboxylic acid-2-methyl-4-0x0-3-(2propenyl)-2-cyclopenten1-yl-ester) was from Dr. I. Yamamoto, Tokyo University of Agriculture, Tokyo, Japan; DDT (l,l, ltrichloro-2,2-bis(p-chlorophenyl)ethane, DDE (1,l -dichloro-2,2-bis(p-chlorophenyl)ethene, and parathion (O,O-diethylO-p-nitrophenyl phosphorothioate) were purchased from Chem Services, West Chester, Pennsylvania. L-[7,8-3H]norepinephrine (r3H]NE,* 30-40 Ci/mmole) and 45CaC12 (17 mCi/mg) were obtained from 2 Abbreviations nephrine; NSM,
used: [3H]NE, normal superfusion
L-[7,VH]norepimedia; DM,
po-
CLARK
Amersham Radiochemicals, Arlington Heights, Illinois, and New England Nuclear, Boston, Massachusetts, respectively. D600 (5[3,4-dimethoxyphenylethyl)methylamino]- 2 -isopropyl- 2 -(3,4,5trimethoxyphenyl)valeronitrile hydrochloride) and D595 (5-[(3,4-dimethoxyphenylethyl)methylaminol-2-(3,4-dichlorophenyl)-2-isopropylvaleronitrile hydrochloride) were gifts of Professor Kretzschmar and Professor Oberdorf, Knoll AG, West Germany, respectively. All other organic and inorganic chemicals were obtained from Sigma Chemical Co., St. Louis, Missouri. Preparation of synaptosomes. Synaptosomes were prepared from whole brains by the method of Whittaker et al. (13) as modified by Hajos (14). Male rats were sacrificed by cervical dislocation and whole brains were removed. Brain matter with a wet weight of approximately 3-4 g was disrupted using a Teflon-glass homogenizer in 20.0 ml 0.32 M sucrose and then centrifuged. The P, fraction (crude mitochondrial fraction) was resuspended in 5.0 ml 0.32 M sucrose, layered onto a discontinuous sucrose density gradient of 0.32, 0.8, and 1.2 M sucrose, and centrifuged at 50,OOOg for I hr. The material at the 0.8- 1.2 M interface was collected by aspiration. This was returned to a more physiological normotonic environment by addition of small volumes of ice-cold normal superfusion media (NSM, Table 1) over a 30-min period to a final volume equal to four times the collected synaptosomal volume (15). The equilibrated synaptosomes were then pelleted at 15,OOOg for 10 min and resuspended in 2.0 ml ice-cold NSM. The concentration of protein was set typically between 8 and 11 mg/ml (16). tassium-depolarizing media: OCaNSM, zero-calcium normal superfusion media: OCaDM, zero-calcium potassium-depolarizing media; LCNSM, low-calcium normal superfusion media: LCDM, low-calcium depolarizing media; D600, S-[(3.4-dimethoxyphenylethyl)methylamino]-2-isopropyl-2-(3.4,5-trimethoxyphenyl)valeronitrile hydrochloride; D595 5-[(3,4-dimethyloxyphenylethyl)methylamino]-2-(3.4-dichlorophenyl~L-isopropyl valeronitrile hydrochloride.
NOREPINEPHRINE TABLE
RELEASE
BY ALPHA
I
Representative Buffer Compositions of External Solutions0 (mmoles/Liter) Solutic& NSM DM OCaNSM OCaDM LCNSM LCDM
N&l 128 77 128 77 128 77
KC1
D-Ghcose
5 56 5 56 5 56
16 16 16 16 17 17
CaCl,
M&I,
I 1 0.01 0.01
I I -
Ba’&
-
* In addition, all solutions contained 12 ~,44 nialamide and 20 mM Hepes. All solutions were adjusted to pH 7.35 with Tris base. * NSM, normal superfusion media; DM. depolarization media; OCa-. zero-calcium buffers; LC-. low-calcium buffers.
Uptake and release of [3H]NE. The procedure for uptake and release of 13HlNE was identical to that developed by Levi and Raiteri (17) as modified by West and Fillenz (18). Synaptosomal aliquots (0.1 ml) were incubated at 37°C with 0.139 kiI4 [3H]NE (approx. 111, 446 cpm per aliquot) for 15 min with constant shaking. Uptake was terminated by addition of 1.0 ml icecold NSM. Background radiation was determined from blanks which consisted of identical incubations carried out at 0°C. All solutions were filtered with suction onto 0.65-km cellulose acetate filters (Millipore Co., Bedford, MA) and washed with 10 ml of ice-cold NSM to remove any unbound [3H]NE. Uptake of [3H]NE was determined by extracting filters overnight in I M perchloric acid (1.0 ml) and measuring the extracted (acid-solubilized aliquot) and nonextracted (filter) by liquid scintillation spectrometry. Specific uptake was determined by subtracting the total cpm value of the 0°C incubation from that which occurred at 37°C. Release of [3H]NE was measured using a continuous perfusion device that consisted of eight IO-ml polypropylene syringes fixed in a container which was maintained at 37 ? 2°C. Synaptosomal aliquots on cellulose acetate filters were housed in 25-mm Swinnex propylene filter holders (Millipore Co.) which were attached to the bottom of each syringe barrel. Tygon tubing carried perfused liquid from filters to scintillation vials via peristaltic pumps. There existed a
CYAN0
PYRETHROIDS
129
3-ml dead volume in the tubing. Hence at a flow rate of 0.5 ml/min, it would take fluid approximately 6 min to appear in the scintillation vial. Twenty-four OS-ml fractions were collected over a 24-min period from each barrel. At fraction 6 (6 min), selected barrels were depolarized by addition of 2.0 ml KC depolarizing media (DM, Table I). Depolarization was terminated after 3 min by diluting the remaining solution IO-fold by addition of NSM. The radioactivity (cpm t3H]NE) in each fraction was determined by liquid scintillation spectrometry. Calculation of [3HjNE released. Release of [3H]NE was expressed either as a fractional rate constant or as a summation of fractional rate constant differences. The fractional rate constant is the amount of [3H]NE released in each I-min fraction (i.e., 0.5 ml) as a percentage of the radioactivity remaining in synaptosomes during the preceding minute (19-22). After determining fractional rate constants for all barrels, a fractional average of all fractional rate constants from similarly treated barrels was calculated. The fractional average determined for nondepolarized barrels was then subtracted from the fractional average determined for depolarized barrels to give a fractional average difference. The fractional average differences for fractions 12 through 20 were summed and these summations of fractional average differences (e.g., summations of [3H]NE released) were used to compare the effects of various insecticides and inhibitors. Addition
of inhibitors
and insecticides.
In experiments which used water-insoluble inhibitors or insecticides, compounds were solubilized in 95% ethanol. Their amounts were adjusted to give a final assay concentration when 1 .O ~1 was added to a 0. l-ml synaptosomal aliquot. Synaptosomes were preequilibrated for 10 min on ice prior to the addition of [3H]NE. Ethanol was included in all control tubes and accounted for not more than 1% of the total volume. Tetrodotoxin was solubilized in 6.0 x 10m3 M sodium citrate (pH = 4.8). Sodium ci-
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AND
trate solution was added to control tubes (assay concentration was 6 x lop5 M>. Tetraethylammonium was solubilized in distilled water. In experiments with insecticides, incubation tubes were coated with Carbowax PEG 20000. Insecticides were added to carbowax-treated tubes and incubated under standard conditions. Solutions were transferred to noncoated tubes prior to loading with [3H]NE. This transfer was necessary in that carbowax (polyethylene glycol) readily absorbs amines such as norepinephrine. Calcium-45 uptake studies. Synaptosomes were preincubated with either deltamethrin or 1.0 ~1 of 95% ethanol prior to addition of unlabeled norepinephrine (0.139 kiV NE). The incubation with unlabeled NE was performed as described for [3H]NE. Uptake of unlabeled NE was terminated after 15 min by addition of 10.0 ml ice-cold low-calcium normal superfusion media (LCNSM, Table 1). Aliquots were filtered with suction onto 0.65~km Millipore filters and perfused as described with an additional 3.0 ml of LCNSM. Selected barrels received 2.0 ml of low-calcium depolarization media (LCDM) containing 0.1 &i 45CaCl,/ml (approx. 250,000 cpm). The remaining barrels received LCNSM which also contained 0.1 uCi 45CaCl,/ml. After a 3-min 45Ca perfusion period, all filters were washed with 15.0 ml ice-cold LCNSM and the 45Ca present on filters was determined by liquid scintillation spectrometry. Statistics. T tests and least-squares regression analyses were performed on the cyber CDC mainframe computer located at the University of Massachusetts using the Statistical Package for the Social Sciences (SPSS). RESULTS
Characterization of synaptosomal uptake of r3aNE. Uptake of [3H]NE is carried out by two distinct mechanisms: a high affinity-active transport mechanism known as uptake 1 (24) which is ouabain sensitive
CLARK
and dependent on the presence of external Na+, and a low-affinity transport system known as uptake 2 (25) which is ouabain insensitive and less dependent on external Na+. Table 2 shows that both of these uptake mechanisms are functional in the present preparation. Uptake of [3H]NE in the presence of ouabain (10e3 M> was inhibited 60 ? 4% when compared with control values. When Na+ was replaced by choline on an equimolar basis, uptake was inhibited 64 + 3% when compared with control values. Therefore, approximately 60% of [3H]NE taken up by synaptosomes was by the high-affinity uptake 1 mechanism. The remaining 40% of uptake was ouabain insensitive and independent of external Na+ concentration which is in agreement with the uptake 2 mechanism. The average uptake to [3H]NE for these experiments was 3.82 2 0.59 pmoles/mg protein/ 15 min. The remaining results summarized in Table 2 reveal that modification of ion fluxes has little effect on [3H]NE uptake. Addition of the K+ channel blocker, tetraethylammonium, the Cl- channel blocker, picrotoxinin, or the Ca2+ channel blockers, D600 and D595, had no effect on synaptosomal uptake of [3H]NE. Effect of insecticides on synaptosomal uptake of [3H]NE. Pyrethroids had little or no significant (P < 0.05) effect on uptake of [3H]NE by synaptosomes at concentrations of lop5 M or less (Table 3). Of the pyrethroids tested at 1O-5 M, only fenvalerate resulted in a significant inhibition of [3H]NE uptake but reduction was only 11% of the control value. The organochlorine, DDT, was the most inhibitory insecticide tested, resulting in a 21% inhibition of control [3H]NE uptake at 10e6 M. Neither DDE nor parathion had any significant effect on synaptosomal uptake of [3H]NE at concentrations up to 10e4 M. Characterization of synaptosomal release of [3HjNE. Functionally intact synaptosomes can be artificially depolarized by increasing external concentrations of K+
NOREPINEPHRINE
RELEASE
BY ALPHA
CYAN0
131
PYRETHROIDS
TABLE 2 Effect
of Biochemical
Inhibitors
on [‘H]NE
Uptake
into Rat Brain
Synaptosomes
[3H]NE uptake Concentrationa
Inhibitor
Ouabain Choline Tetrodotoxin Tetraethylammonium Picrotoxinin D600 D595
Control
(pmoles [3H]NE + SEMlmg protein/l5 min) 4.45 t 0.05 1.78 2 4.45 t 0.05 1.62 2 3.32 ” 0.23 3.71 k 3.32 r 0.23 3.53 k 3.43 r 0.30 4.25 f 4.45 t 0.05 5.00 k 3.32 -c 0.23 3.52 2
10m3 M 128 x 10m3M lo-‘M
5 x 1O-3 M 2 x 1O-5 M IO-*
Treated
M
1O-5 M
O.l6*J’ 0.11” 0.18 0.20 0.38 0.46 0.01
N = 3’ N=3 N=2 N=3 N=2 N=3 N=2
u Control synaptosomes were treated with 1.O ~195% ethanol. Treated synaptosomes received the concentration of inhibitor as listed in 1.O pl 95% ethanol. b Means followed by an asterisk are significantly (P < 0.05) different from control means (one-tailed t test). c N denotes number of experiments. Each experiment contained four replicates.
ions which in the presence of Ca*+ leads to release of vesicle-bound neurotransmitters (18, 26,27). When viable synaptosomes are continuously perfused and pulsed by depolarized K+ in the presence of Ca*+. they release neurotransmitter in a measurable and reproducible pattern (2, 8). The data in Fig. 1 demonstrate that rat brain synaptosomes utilized in this work are viable and reproduce a similar pattern of neurotrans-
mitter (e.g., [3H]NE) release under the present experimental conditions. Only synaptosomes in the presence of Ca2+ showed an enhanced rate of t3H]NE release when K+ depolarized (Fig. IA). This is illustrated by the increase in fractional rate constants for fractions (min) 12 through 20, inclusively. Synaptosomes which were perfused in media containing Mg2+ instead of Ca2+ (open circles) or which never re-
TABLE Effect
of Insecticides (pmol
on [3HjNE Uptake [3HjNE ? SEMimg
3 into Rat Brain Synaptosomes protein/l5 min)
[3H]NE uptake Insecticide
Control”
1O-6 M
1O-5 M
1O-4 M
Deltamethrin Fenvalerate Cypermethrin des-Cyano Deltamethrin des-Cyano Fenvalerate Allethrin Permethrin DDT DDE Parathion
3.44 k 0.19 3.24 k 0.47 3.23 k 0.13
3.42 r 0.15 3.04 * 0.14 3.48 2 0.13
3.66 -e 0.37 2.87 k 0.06* 3.47 f 0.22
2.53 i 0.19*.b 2.13 2 0.26* 3.30 + 0.12
3.43 2 0.17 3.43 3.43 4.02 3.45 3.23 3.23
k k -c f 2 k
0.17 0.07 0.16 0.09 0.03 0.03
4.55 k 0.07 3.19 4.71 2.71 3.05 3.36
2 2 t 2 k
0.14 0.07 0.09* 0.02 0.16
4.73 3.49 4.64 3.00 3.11 3.83
2 + L + k i
0.06 0.24 0.15 0.1s* 0.31 0.09
N = 4’ N=3 N=3 N=3
2.66 3.94 2.28 3.19 3.06
f 2 2 i 2
0.30* 0.27 0.13* 0.09 0.13
N=3 N=5 N=3 N=3 N=3 N=3
a Control synaptosomes were treated with 1.0 ~195% ethanol. b Means followed by an asterisk are significantly different from control means (P < 0.05, one-tailed t test). c N represents number of experiments. Each experiment consisted of four replicates.
132
BROOKS
AND
CLARK 12lo-
;;
A
+ D’ta/ I. Control 8
b
-depolarized
non-depolarized
IL’
6
2
4
6
B
10 12
14 16 1E 20 22
24
FractiondMid FIG. 1. Effect of calcium on the release of [3HjNE. Synaptosmal aliquots M’ere loaded Jc*ith 0.139 (IM 13HlNE in media containing 1 mM Ca2+ and subsequently tc,ashed and perfused wjith media containing either 1 mM Ca2+ (NSM. closed circles) or 1 mM Mg2+ (OCaNSM, open circles). (A) All synuptosomnl aliquots were depolarized ufter 6 min (jiiaction 6, as indicated by arrow) by addition qf media containing 56 mM K+ and either Ca2+ or Mg2+ (DM or OCaDM, respectitaely) for 3 min. (B) The K+ level remained at 5 mM throughout the perfksion (i.e.. nondepolarized). EfJux MWS expressed as a ,fructional rate constant. The fractional rate constants are determined by dividing cpm released during that fraction by the cpm present in the synaptosomal aliquot ut the beginning of the fraction. The results represent an coverage of four experiments titith each experiment consisting of four replicates.
ceived K+-depolarizing media (Fig. lB), regardless of the presence of Ca2+, did not change their rates of [3H]NE release. Under these experimental conditions, the average calcium-dependent, potassiumstimulated release of [3H]NE was calculated to be 70 + 12 fmoles [3H]NE/mg protein/3 min. This value represents approximately 1.8% of total [3H]NE uptake. Effect
of deltamethrin
on [3H]NE
re-
lease. Figure 2 shows the fractional rate constants calculated for [3H]NE release from synaptosomes treated with lop5 M of either deltamethrin (Dlta, open circles) or ethanol (Control, closed circles). Addition of K+-depolarization media to delta-
FractiondMin) FIG. 2. Effect of deltamethrin on [3H]NE efflux. Synaptosomal aliquots were incubated with either lo-= M deltamethrin (Dlta. open circles) or I.0 ~195% ethanol (control, closed circles) prior to loading with 0.139 PM [3H]NE. Aliquots were subsequently naashed and perfked with NSM. (A) Synuptosomes were depolarized by addition of DM for 3 min (us indicated by arrow). (B) Synuptosomes were treated tixith NSM for the entire assay. Results are expressed as ,fractionul rate constants (see Fig. I ). The results represent an average of four experiments nsith each experiment consisting offour replicutes.
methrin-treated synaptosomes produced a large initial spike of [3H]NE release compared with ethanol-treated synaptosomes (Fig. 2A). The initial enhanced spike following K+ depolarization suggests that deltamethrin facilitates Ca2+ entry into the cytosol (22). Furthermore, depolarization of deltamethrin-treated synaptosomes produced a prolonged tailing effect of [3H]NE release which continued through fraction 20. Enhanced release of [3H]NE was not evident in synaptosomes which did not receive K+ depolarization regardless of the presence of deltamethrin or ethanol (Fig. 2B). Thus, increased [3H]NE release due to the addition of deltamethrin was evident only during depolarization and in the combined presence of Ca2+. As illustrated in Fig. 3, the effect of deltamethrin is dose dependent. Synapto-
NOREPINEPHRINE
RELEASE
BY ALPHA
to-i0 Deltamethrln
CYAN0
10-g Concentration
133
PYRETHROIDS
10-7
10-s CM)
FIG. 3. Effect of various concentrations of deltamethrin on release of [‘HINE. Synaptosomal fractions were incubated toith either deltamethrin (crossed bars) or I .O $957~ ethanol (open bars) prior to loading with 0.139 FM [3HjNE. Aliquots were subsequently washed and perfused tijith NSM. After 6 min (fraction 6), selected aliquots were depolarized with DM for 3 min. Results are expressed as a summation of [‘HjNE released (see Materials and Methods section). Both nondepolarized and depolarized aliquots received similar treatments (i.e., either all received deltamethrin or all received 95% ethanol). The results represent an average of two experiments k standard error measurement (SEM). Each experiment consisted offour replicates.
somes treated with deltamethrin at lo-” and lo-lo M gave [3H]NE release values (i.e., summation of [3H]NE released) upon depolarization that were not significantly different (P < 0.05) from control values (13.6 L 0.1% for lo-lo A4 compared with 13.7 i 2.3% for controls, and 12.9 ? 2.0 for lo-” M compared with 14.7 + 1.0% for controls). Enhanced [3H]NE release apparent at lop9 M deltamethrin was significantly different from control values (P < 0.05) (treated synaptosomes released 13.2 L 0.3% compared with 9.7 ~fr.0.2% for controls). Maximum stimulation was achieved at lo-’ M deltamethrin (treated synaptosomes released 27.8 + 3.7% compared with 12.6 ? 0.5% for controls) and this response became saturated at higher concentrations of deltamethrin. The average enhanced release of [3H]NE in the presence of lo-’ M deltamethrin for these experiments was calculated to be 115 2 7.1 fmoles [3H]NE/mg protein/3 min which
represents approximately 3.01% of the total [3H]NE load. Effect of deltamethrin on 45Ca uptake. Since norepinephrine release has been demonstrated to be highly dependent on Ca*+ influx, an attempt was made to directly measure the effect of deltamethrin on Ca*+ uptake. As illustrated in Fig. 4, deltamethrin enhanced synaptosomal uptake of 45Ca during K+ depolarization in a dose-dependent manner (crossed bar) over control values (solid bar). Significant enhancement (P < 0.05) was noted with deltamethrin concentrations greater than lo-lo M. Potassium-stimulated 45Ca uptake reached a maximum enhanced level at a deltamethrin concentration of lop7 M (550 ? 50 fmoles 45Ca/mg protein/3 min) with saturation noted at lop5 M. The control value of K+-stimulated 45Ca uptake for these experiments was calculated to be 250 i 55 fmoles 45Ca/mg protein/3 min. To correlate the effect of deltamethrin on
134
BROOKS
Deltamethrin
AND
CLARK
Concentration
(Ml
4. Effect of deltamethrin on %a uptake. Synaptosomal aiiquots tizere incubated JO min M’itJl either deltamethrin or I.0 ~1 95% ethanol. Synaptosomes were loaded with 0.139 )LM unlabeled NE, washed, and perfused with NSM. After 6 min (fraction 6). selected fractions were depolarized by the addition of 2.0 ml of LCDM containing 0.1 PCi of 4sCa per milliliter (solid plus crossed bars). The remaining aliquots received 2.0 ml of LCNSM containing 0.1 @i of “Y?a per milliliter (open bars). After 3 min, all synaptosomal aliquots were washed with 15.0 ml LCNSM, and the radioactivity remaining on the filters teas determined by liquid scintillation spectrometty. Solid burs represent the amount of ‘Yu tuken up bv depolarized synaptosomes which Msere preincubated with 95% ethanol. Crossed bars represent any additional amount of ‘Vu uptake which w’as in excess of the control 1~11~ due to the presence of deltamethrin. The mean values are abarrages & SEM of two experiments, euch consisting of four replicates. FIG.
[3H]NE release and 45Ca uptake, enhanced values of [3H]NE release (closed circles) and 45Ca uptake (open circles) due to increasing the concentration of deltamethrin from lo-lo to IO-’ M were subjected to a regression analysis and co-plotted (Fig. 5). The effective dose of deltamethrin which resulted in a 50% maximal response (i.e., ED,,) for both these phenomena was estimated from these regressed lines. In the case of enhanced [3H]NE release (solid line), the ED,, for deltamethrin was calculated to be 2.9 x 1O-9 M compared with 2.4 x 10m9 M for enhanced 45Ca uptake (dashed line). The apparent stoichiometry between 45Ca uptake and [3H]NE release estimated from these lines is approximately 5.8 Ca2+ to 1 NE. Comparison of the effect of various in-
secticides on [3HJNE release. As judged by the summation value of [3H]NE released for fractions 12 to 20, deltamethrin was the most effective compound in enhancing L3H]NE release. Deltamethrin enhanced 13H]NE release 114% compared with an untreated control value (Fig. 6A). Other alpha cyano pyrethroids were also effective in enhancing release. Cypermethrin resulted in a 64% enhancement while fenvalerate enhanced [3H]NE release 40% above the control value. The relative ability of deltamethrin, cypermethrin, and fenvalerate to enhance [3H]NE release is calculated as 1:0.56:0.35, respectively. This ratio correlates well with the relative toxicity ratio calculated from the acute oral LD,, values for rat (i.e., deltamethrin = 135 mg/kg, cypermethrin = 251 mg/kg, and
NOREPINEPHRINE
RELEASE
Deltamethrin
BY ALPHA
Concentration
CYAN0
PYRETHROIDS
135
(M)
FIG. 5. Least-squures regression analysis of deltamethrin-stimulated [‘H]NE release (@) and 4SCa uptake (0). Using the same experimental protocol as detailed in Figs. 3 and 4, respectively, six values of enhanced [3HJNE release and 4sCa uptake at various concentrations of deltamethrin (from lO&‘O to lo-’ M) were regressed andplotted. The regression analysis for enhanced [3H]NE release (-) dae to the addition of deltamethrin gave an rz value of 0.82, and 0.98 f or 45Ca uptake (--). Each data point represents the mean of 01’0 experiments which consisted offoar replicates each.
fenvalerate = 451 mg/kg) (30) which results in a ratio of 1:0.54:0.30. Pyrethroids that lacked an alpha cyano grouping were greatly reduced in their ability to enhance [3H]NE release (Fig. 6B). Addition of bioallethrin resulted in a 4.7% enhancement and des-cyano cypermethrin in a 1.2% enhancement but neither was significantly different (P < 0.05) from control values. Des-cyan0 deltamethrin and des-cyano fenvalerate were also without an effect. Table 4 summarizes the effect of DDT, DDE, and parathion on [3H]NE release during K” depolarization. At 10es M, DDT produced a highly significant (P < 0.05) effect resulting in a 92% inhibition of [3H]NE release compared with controls. At the concentration, however, DDT has been determined to significantly inhibit synaptosomal [3H]NE uptake (see Table 3). Neither parathion nor DDE at concentrations of
lops M had any significant (P < 0.05) effect on synaptosomal efflux of [3H]NE. DISCUSSION
Enhanced release of [3H]NE from rat brain synaptosomes by alpha cyano pyrethroids is evident only in the combined presence of external Ca*+ and membrane depolarization. This indicates that insecticides like deltamethrin act as use-dependent compounds (31-34). Because release of [3H]NE is highly dependent on Ca*+ entering the cytosol of the synaptosome, it can be assumed that deltamethrin may act on entities which transport Ca*+, such as the voltage-sensitive sodium and calcium channels (29, 35). In a similar study, [3H]NE was released from rat brain cortex slices by electrical stimulation. This release was shown to be absolutely dependent on extracellular calcium and was blocked not only by tetrodotoxin but also by the cal-
BROOKS
136
AND
CLARK
FIG. 6. Effect of various pyrethroid insecticides on release of [3HjNE. Synaptosomal aliqaots were incubated with either a 10m5 M concentration ofpyrethroid (crossed bars) or 1.0 ~195% ethanol (open bars) prior to loading with 0.139 @I [3H]NE. Aliquots were subsequently washed and perfused with NSM. After 6 min (fraction 6), selected aliquots were depolarized with DM. (A) The effects of alpha cyano pyrethroids (type II). (B) The effects of pyrethroids lacking such a moiety (type I). Results are expressed as a summation of [‘HjNE released -t SEM (see Materials and Methods section). Summations expressed are an average sf ttijo experiments, each consisting offour replicates.
cium channel antagonist, D600, and by manganese (22). Ghiasuddin and Soderlund (32) have determined a Kd for [3H]descyano deltamethrin binding to mouse brain synaptosomes of 4 x lop8 M. Because del-
Effeecr of Nonpyrethroid
Insecticides K+-Depolarized
[3H]NE Insecticide”
DDT DDE Parathion
TABLE 4 on Ca 2f-Dependent Synaptosomes
a The concentration of all insecticides was * Control synaptosomes were treated with c Relative insecticide-induced NE release of control value. d Values represent means of two experiments asterisk are significantly (P < 0.05) different
Treated release
[3HJNE
Release
? SEM)d 1.2 ? 2.4* 14.1 + 1.3 12.5 2 1.3
10m5 M. 1 .O ~1 95% ethanol. is the r3H]NE released
from
Relative insecticide-induced [3H]NE release (percentage of control released)c
release
Control* (summation [3H]NE 15.4 & 0.8 15.2 * 1.8 14.5 * 2.1
tamethrin is 10 times more toxic, it can be assumed that it is also 10 times more potent and hence has 10 times greater affinity for its receptor. If this assumption is true, deltamethrin’s apparent Kd should be in the
7.8 92.7 86.2
from
treated
synaptosomes
with four replicates for each insecticide. from control means (one-tailed t test).
Means
as a percentage followed
by an
NOREPINEPHRINE
RELEASE
BY ALPHA
order of 4 x 10e9 M. This compares well with the ED,, of deltamethrin of approximately 3 x 1O-9 M which was calculated for both [3H]NE release and 45Ca uptake in this study. Electroencephalographic studies have shown that alpha cyano pyrethroids exert a highly selective action at the caudate nucleus in the corpus striatum of the brain (36). The increased caudate blood flow caused by intraperitoneal administration of deltamethrin preceded the development of electroencephalogram spike discharges and the development of motor symptoms typical of type II pyrethroids. Cismethrin. a type I pyrethroid, did not produce enhanced blood flow in the caudate nucleus. Interestingly, brain arterioles and capillaries are innervated by adrenergic nerve fibers indicative of a central adrenergic control of cerebral blood flow (37). These results correlate well with those presented initially by Aldridge et al. (38), who showed that acetylcholine was significantly depleted from rat brain after oral administration of deltamethrin. Cismethrin was greatly reduced in its ability to deplete this rictu-otransmitter while DDT resulted in no depletion whatsoever. Recently, it has been shown that only the toxic s-acid, s-alcohol isomer of fenvalerate invoked an enhanced neurotransmitter release in rabbit brain striatal slices (39). A similar enhanced neurotransmitter release was not apparent in rabbit brain hippocampal slices. In related studies that examined the effects of several pyrethroids and DDT on insect neuromuscular preparations, it was reported that alpha cyano pyrethroids were very potent in increasing the rate of miniature excitatory postsynaptic potentials. DDT and non-alpha-cyan0 pyrethroids were much reduced in their ability to increase the rate of potential firing (40). Recently, deltamethrin has been shown to cause almost complete depletion of synaptic vesicles from presynaptic motor nerve terminals of the house fly. This indi-
CYAN0
PYRETHROIDS
137
cates a presynaptic action of deltamethrin (41). Interestingly, mitochondria of treated terminals were reported to be swollen and have vacuolated interiors. It is well established that mitochondria function as a major calcium regulator within presynaptic nerve terminals (15). One possible explanation which links the effect of deltamethrin on [3H]NE release to pyrethroid symptomology is that by causing the synaptosome to rapidly lose [3H]NE, the nerve terminal is depleted of NE stores. Reduction of NE in the brain results in loss of blood vessel constriction and ultimately in increased blood flow. Depletion of brain NE stores has been shown to be highly correlated to the convulsive state (9, 1I), a condition evident in deltamethrin poisoning. Finally, compounds which have been shown to deplete monoaminergic neurotransmitters like norepinephrine significantly potentiate the toxicity of pyrethroids (6). Thus, there appears to be a correlation of enhanced [3H]NE release, depletion of brain stores of NE, increased cerebral blood flow, and the convulsive state, all of which are caused by the action of deltamethrin. These results are in accordance with the theory that voltagesensitive ionic channels in nerve cells are the primary target of pyrethroids (3). As shown by Vijverberg and de Weille (42). the most pronounced effect of alpha cyano pyrethroids on nerve cells under voltageclamp conditions is the prolongation of the time constants calculated for the decay of sodium tail currents. Most notable is the fact that all alpha cyano pyrethroids result in greatly enhanced time constants reflecting extremely slow kinetics of the tail currents. The addition of an alpha cyano grouping to lR-trans-permethrin (which yields lR-trans-cu-S-cypermethrin) caused the time constant to increase from 7.3 to 1 I14 msec, respectively. Prolonged opening of voltage-sensitive ionic channels, both sodium and calcium, could enhance calcium influx and result in enhanced neu-
138
BROOKS AND CLARK
rotransmitter release, and ultimately in the depletion of synaptic vesicles and neurotransmitters in presynaptic nerve terminals. Indeed, our data on enhancement of norepinephrine release by alpha cyano pyrethroids (i.e., deltamethrin 114%, cypermethrin 64%, and fenvalerate 40% over control release) when converted to a relative ratio (e.g., 1:0.56:0.35) compares well with Vijverberg and de Weille’s data on time constant enhancement by alpha cyano pyrethroids (i.e., 1772: 1114:603 msec of 1:0.63:0.34, respectively). It is not expected that non-alpha-cyan0 pyrethroids would be as effective in creating a prolonged depolarization as are the alpha cyano pyrethroids.
9.
10.
11.
12.
13. ACKNOWLEDGMENTS This work was supported by research Grant RR07048-19, NIH-BRSG, and by the Massachusetts Agricultural Experimental Station, UMASS, Amherst. Massachusetts. REFERENCES 1. R. D. Verschoyle and W. N. Aldridge. Structureactivity relationships of some pyrethroids in rats, Arch. Toxicol. 45, 325 (1980). 2. D. E. Ray, An EEG investigation of decamethrin induced choreoathetosis in the rat, Exp. Brain Res. 38, 221 (1980). 3. J. Rickard and M. Brodie, Correlation of blood and brain levels of the neurotoxic pyrethroid deltamethrin with the onset of symptoms in rats, Pestic. Biochem. Physiol. 23, 143 (1985). 4. D. E. Ray and J. E. Cremer. The action of decamethrin (a synthetic pyrethroid) on the rat, Pestic. Biochem. Physiol. 10, 333 (1979). 5. J. E. Cremer, V. J. Cunningham, D. E. Ray, and G. S. Sarna. Regional changes in brain glucose utilization in rats given a pyrethroid insecticide, Brain Res. 194, 278 (1980). 6. C. G. Staatz, A. S. Bloom, and J. J. Lech. A pharmacologic study of pyrethroid neurotoxicity in mice, Pestic. Biochem. Physiol. 17, 287 (1982). 7. C. G. Staatz-Benson and M. J. Hosko. Interaction of pyrethroids and mammalian spinal neurons, Pestic. Biochem. Physiol. 25, 19 (1986). 8. A. D. Rudzik and G. A. Johnson, Effect of amphetamine and amphetamine analogs on convulsive thresholds, in “International Symposium on Amphetamines and Related Com-
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14. F. Hajos. An improved method for the preparation of synaptosomal fractions in high purity, Brain Res. 93, 485 (1975). 15. M. P. Blaustein, R. W. Ratzlaff, and N. K. Kendrick. The regulation of intracellular calcium and presynaptic nerve terminals, Ann. N. Y. Acad. Sci. 307, 195 (1978). 16. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. M. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 17. G. Levi and M. Raiteri, Synaptosomal transport processes, Int. Ret,. Neurobiol. 19, 51 (1976). 18. D. P. West and M. Fillenz, Storage and release of noradrenaline in hypothalamic synaptosomes, J. Neurochem. 3X6), 1323 (1980). 19. F. Orrego, J. Jankelevich, C. Ceruti, and E. Ferrera, Differential effects of electrical stimulation on release of 3H-noradrenaline and i4Caminoisobutyrate from brain slices, Nature (London) 251, 55 (1974). 20. F. Orrego and R. Miranda, Electrically induced release of 3H-GABA from neocortical thin slices, J. Neurochem. 26, 1033 (1976). 21. G. Levi, V. Gallo, and M. Raiteri, A reevaluation of veratradine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings, Neurochem. Res. X3), 281 (1980). 22. F. Orrego and S. Sanchez-Armass, Electrically induced release of 3H-NE from rat brain cortex slices: A kinetic analysis of the dependence of
NOREPINEPHRINE
RELEASE
BY ALPHA
extracellular
calcium, Pharmacol. Res. 13(10), 949 (1981). D. W. Helmuth, S. M. Ghiasuddin, and D. M. Soderlund, Poly(ethylene glycol) pretreatment reduces pyrethroid adsorption to glass surfaces, j. Agric. Food Chem. 31, 1127 (1983). L. L. Iverson, Role of transmitter uptake mechanisms in synaptic transmission, &it. J. Pharmacol. 41, 571 (1971). .I. S. Gillespie. Uptake of noradrenaline by smooth muscle, Brit. Med. BulL. 29, 136 (1973). M. P. Blaustein, Effects of potassium, veratradine, and scorpion venom on calcium accumulation and transmitter release by nerve terminals in vitro, /. Physiol. 247, 617 (1975). G. Maura, A. Pittaluga. A. Ricchetti. and M. Raiteri, Noradrenaline uptake inhibitors do not reduce the presynaptic action of clonidine on 3Hnoradrenaline release in superfused synaptosomes, Naunyn-Schmied Arch. Pharmacol. 327, 86 (1984). M. Raiteri, F. Angelini, and G. Levi, A simple apparatus for studying the release of neurotransmitters from synaptosomes. Eur. J. Phnrmacol. 25, 411 (1974). R. R. Llinas, Calcium in synaptic transmitter, Sci. Amer. 287(4), 56 (1982). C. R. Worthing, “The Pesticide Manual,” 7th ed.. British Crop Protection Council, U.K., 1983. A. E. Lund and T. Narahashi, Kinetics of sodium channel modifications as the basis for the variation in the nerve membrane effects of pyrethroids and DDT analogs, Pestic. Biochem. Common.
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42.
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