Promotion of norepinephrine release and inhibition of calcium uptake by pyrethroids in rat brain synaptosomes

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

29, 187- 196 (1987)

Promotion of Norepinephrine Uptake by Pyrethroids

Release and Inhibition of Calcium in Rat Brain Synaptosomesl

JOHN D. DOHERTY,~ KEIICHIRONISHIMURA,

NORIO KURIHARA,* ANDTOSHIO FUJITA~

Department of Agricultural

Chemistry and *Radioisotope Research Center, Kyoto University, Kyoto 606, Japan Received February 16, 1987; accepted September 9. 1987

A series of 25 pyrethroids were assessed for their effects on Na+-dependent norepinephrine release and on CaZ+ uptake in vitro using a crude rat brain synaptosomal preparation. The most effective pyrethroids required a concentration of 3-10 (*M to promote norepinephrine release. Plotting release data versus lipophilicity (as log P) for each compound resulted in a parabolic curve with log P,,p, being 5.4 for maximal release. The release promoted by most of the compounds assessed at 30 p&I could not be or was only partially reversed by either tetrodotoxin or substituting choline for Na+ conditions which readily reversed the release promoting effects of veratridine. Thus, many pyrethroids, particularly those without the a-cyan0 group, did not display their expected effects on the Na+ channel in rat brain. When assessed at 5 )LM, pyrethroids inhibited, had no effect, or caused increases in the amount of Ca2+ incorporated in the presence of ATP. The effectiveness of the various pyrethroids to inhibit Ca2+ uptake again displayed a parabolic relationship with log Popt being 6.4. It was concluded that the variations in pyrethroid effects on norepinephrine release and Ca*+ uptake are not solely related to their particular chemical structures, but to lipophilicity. The effects of many pyrethroids on CaZ+ metabolism, particularly displacement of bound Ca2+, better explain the transmitter release promoting properties in vitro rather than a direct effect on the Na+ channel. No direct relationship between known toxicity to mammals and Ca’+ inhibition by pyrethroids was established. 0 1987 Academic Press. Inc

INTRODUCTION

As a chemical class, pyrethroids are extremely toxic to the insect nervous system and their locus of action in insects is widely accepted to be on the Na+ conductance channel (1, 2). These agents affect the Na+ channel in axons in such a manner as to allow an increased influx of Na+ which in turn causes unstable nerves and subsequent neurophysiological and neurotoxicological responses. Pyrethroids would be expected to have this same phenomenon in the synaptic region. To assess this hypothesis, veratridine-induced release of preloaded transmitters can be used as a model system. Veratridine promotes the release of preloaded GABA and norepiL Quantitative Structure-Activity Studies of Pyrethroids, Part 11. * On leave from the Environmental Protection Agency, Washington, DC. 3 To whom correspondence should be addressed.

nephrine in the presence of Na+ by allowing an increased influx of Na+ (3) and this release can be abolished by TTX4 which blocks the influx of Na+ through the Na+ channel (4, 5) and by Ch+ substitution for Na+ . Using the veratridine-induced release of transmitters as a model system, it was demonstrated that resmethrin enhances the release of GABA, dopamine, and norepinephrine from rat brain synaptosomes but this effect of resmethrin is affected by neither TTX nor Ch+ (6). In contrast, the a-cyan0 pyrethroids, deltamethrin and cypermethrin, promoted the release of GABA from rat brain (6) and deltamethrin promoted the release of GABA from guinea pig brain (7) but the effects of these cr-cyano-substituted pyrethroids were readily reversed by TTX. In the rat brain experiments, it was noted 4 Abbreviations used: TTX, tetrodotoxin: choline; GABA, y-aminobutyric acid.

Ch+,

187 0048-3575187 $3.00 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved

188

DOHERTY

that high concentrations of pyrethroid (IO-75 FM) were required to promote the release of transmitters (6). Resmethrin but not deltamethrin was also shown to inhibit the ATP-dependent uptake of Ca*+ by synaptosomes at lower concentrations (Iso: 2.2 PM) than those at which it promoted transmitter release. Since Ca*+ uptake inhibition occurred in the absence of Na+ , an effect of pyrethroids independent of that on the Na+ channel was established. In this paper, the hypothesis that pyrethroids act on the Na+ channel in rat brain synaptosomes was further evaluated in vitro using a set of 25 pyrethroids by assessing for their effects on Na+ dependent norepinephrine release. These same pyrethroids were also assessed for their effects on Ca2+ uptake and displacement of bound Ca2+.

ET AL.

ml of ice-cold assay buffer. The filters were dissolved and counted in Aquasol (New England Nuclear Co.). 45Ca2+ uptake inhibition and displacement. An aliquot of 100 ~1 of the P, fraction containing about 200 p,g of protein was added to 1.9 ml of buffer (3 mM MgCl, and 50 mM Tris-HCl, adjusted to pH 7.4) and 3 ~1 of either methanol alone or a methanol solution of test agent was added and 10 min were allowed for preincubation at 0-4°C. The uptake reaction was started by adding 40 ~1 of a mixture of ATP(Tris) and CaClz (containing 4sCa2+) to give a final concentration of 2 mM ATP and 0.1 mM CaCl, and transferring to a 37°C water bath. After allowing 10 min for the uptake of 45Ca2+ , the mixture was filtered through a 0.45-urn filter (Sartoriusf and washed twice with ice-cold stopping solution (buffer composition as above plus 100 mM NaCl). DisMETHODS AND MATERIALS placement of the bound 45Ca2+ was studied Rat brain samples. Rat brains were ex- by adding 3 ~1 of the test materials in methcised from male Wistar rats (150-300 g). anol to the P, suspension which had been The P2 fraction was obtained from the ho- preloaded for 10 min using the uptake assay mogenate of the forebrain according to the as above and allowing predetermined times method described previously (6). Protein for displacement effects before filtration. analysis was by the method of Lowry (8). The filters were dissolved and counted in Norepinephrine release. The P, material Aquasol (New England Nuclear Co.). This was preloaded with labeled transmitter by method for studying 45Ca2+ uptake and disincubating [3H]norepinephrine in buffer placement was described previously (6. (132 mM NaCl, 5 rnM KCl, 10 mM glucose, 10). 1.2 mM NaH,PO,, 1.2 mM MgCl,. 10 mM Chemicals. 45Ca2+ (as CaCl,) and [3H]Tris-HCl, pH 7.4, and 50 FM pargyline) norepinephrine were obtained from the for 10 min at 37°C (6, 9). After centrifuging New England Nuclear Co., Boston, Masat 17,000g for 10 min, the supernatant was sachusetts. Most of the pyrethroids used discarded and the pellet gently washed with were synthesized in this laboratory (11, 12) sucrose buffer before being resuspended and are listed in Table 1, cis-Permethrin into buffer. The loaded P, material was ali- (16), fenvalerate (20), fenpropathrin (21), quoted (300 ~1 containing 600 pg of protein and cis-cypermethrin (22) were kindly proper assay tube) into 1.7 ml of assay buffer vided by Mr. T. Kadota of the Sumitomo (as above with either 132 mM NaCl or 132 Chemical Co., Osaka. Kadethrin (17) was mM Ch+ chloride) and the release reaction provided by Mr. J. Tessier of the Roussel was started by adding 3 ~1 of test material UCLAF Co., Romainville, France. The in methanol or methanol alone and trans- chemicals 23 (MTI-800: [4-(4-ethoxyferring to a 37°C water bath. The reactions phenyl)-1-(4-fluoro-3-phenoxyphenyl)4were terminated at predetermined times by methylpentanel), 24 (ethofenprox: [2-(4filtering through a 0.45-pm filter (Sar- ethoxyphenyl)-2-methylpropyl 3-phenoxytorius) and were then washed twice with 3 benzyl ether]), and 25 (MTI-790:

PYRETHROIDS

TABLE Promoting

Effects

189

AND RAT BRAIN SYNAPTOSOMES

of Pyrethroids

and Related

1 Compounds

on Norepinephrine

Release”

In Na+ Buffer % release d log 100 - % release Number 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Nameb H o-F o-OPh o-SO&H, m-F m-CH, m-OPr(i) m-OPh m-CN P-W

p-Pen(n) p-SO&H, Cl5

(lR)-trans-Resmethrin ( lR)-trans-Permethrin (lR)-cis-Permethrin ( lR)-cis-E-Kadethrin t lR)-trans-Tetramethrin

Average’ 5 SD%

n

24 If- 5 30 2 6 22 * 1 26 * 9 26 k 0 29 k 5 37 2 8 18 2 6 23 -r- 5 27 2 9 10 k 1 20 * 4 4?2 33 k 5 18 2 6 14 2 5 19 f 4 43 2 6

2 4 2 4 2 2 6 2 2 3 2 3 2 7 2 4 2 6

-0.52 -0.38 -0.57 -0.49 -0.45 -0.40 -0.22 -0.69 -0.54 -0.45 -0.98 -0.61 - 1.49 -0.30 -0.69 -0.65 -0.65 -0.10

16 k 3 12 2 3 31 2 6

3 6 4

-0.73 f 0.07f -0.87 2 0.1Y -0.36 2 0.12f

Obs. k f k ? k f e 2 k t 2 t f 2 2 k t *

Calc. 0.12 0.12 0.02 0.17 0 0.11 0.14 0.19 0.13 0.15 0.04 0.07 0.29f 0.09 0.19 0.14f O.lof 0.14

-0.37 -0.37 -0.65 -0.53 -0.37 -0.39 -0.41 -0.65 -0.38 -0.39 -0.83 -0.53 -1.18 -0.48 -0.61 -0.37 -0.40

In Ch+ Buffer Average’ *SD% 9* 14 15* 11 16& 19 + 24 132 16? 13+ 8 14 0 21* 6 7 8 f 26 +

4 0 6 12 4 3 2

4

n

log P’

2 1 2 1 2 2 1 2 2 2 I I

5.49 5.63 7.57 3.86 5.63 6.05 h.34 7.57 4.92 6.05 8.16 3.86 9.04 6.82 7.44

12 11

5.39 4.73

4

5.85 6.12

(1 R)-trans-a-RS-

Cyphenothrin (15)~cu-S-Fenvalerate c*-RS-Fenpropathrin

- 0.07 -0.39

3+

*

92

2

( 1R)-cis-a-S-

Cypermethrin MT1800 Ethofenprox MTI-790 Veratridine

26 2 6 9?1 24 k 5 928 34 2 8

-0.47 -1.14 -0.50 - 1.21 -0.30

2 ? 2 ? i

O.lY 0.43 0.12 0.46 0.18

&? - 1.24 - 0.49 - 1.17

4 5 2 2+

9.17 6.87 9.03 4

” Each compound was tested at 30 (LM except for compound 13 which was tested at 5 PM. n = number of assays. b Substituents for variously substituted benzyl (lR)-rrans-chrysanthemates are shown for compounds 1-13. See Table 2 in Ref. (17) for structure of compounds 14-25. c The data are expressed as averages ? SD for the number of assays indicated by n. When n = 1 or when only one assay for the condition was available, no SD for the percentage release could be determined. d The percentage release from the individual experiments was first converted to the log value and the average for the log values was determined. The percentage averages listed do not convert to the log values given. The calculated values were derived from Eq. [I]. e Log P values were taken from our previous papers (11,13) and others were calculated according to the additivity principle (14). f Not included in Eq. [l]. g The radioactivity of the test sample was slightly higher than that of the control.

DOHERTY

190

[4-(4-ethoxyphenyl)-1-(3-phenoxyphenyl)4-methylpentane]) were kindly provided by Mr. S. Numata of the Mitsui Toatsu Chemical Co., Tokyo. Veratridine and TTX were obtained from the Sigma Chemical Co., St. Louis, Missouri. RESULTS

Norepinephrine Release

The basic experiments demonstrating the time dependence of norepinephrine release by the P, preparation together with the time course for increased release and the differential effects of TTX on resmethrin and veratridine promoted release were presented previously (6). Pyrethroids were screened for their effectiveness to promote release of norepinephrine in buffer containing either Na+ or Ch+ and the results are presented in Table 1. When release was studied in Ch + buffer, spontaneous release was slower than when studied in Na+ buffer and the pyrethroids appeared weaker in their effects as releasers in Ch+ buffer than in Na+ buffer. Figure 1 shows that the same pyrethroids

ET AL.

which were most effective in releasing norepinephrine in Na+ buffer were also most effective in Ch+ buffer. Tetramethrin (18), compound 7, resmethrin (14), and compound 6 best illustrate the lack of Na+ dependence of norepinephrine release. Veratridine, the positive control for Na+-dependent transmitter release, cis-cypermethrin (22), and fenvalerate (20) were effective in promoting release in Na+ buffer but did not promote release in Ch+ buffer. Several representative pyrethroids which were the strongest releasers were selected and attempts were made to reverse the increase in norepinephrine release by TTX (Table 2). TTX treatment had no effect on the release induced by the most effective releasers, particularly tetramethrin (18), resmethrin (14), and compounds 2 and 7 as TABLE

Norepinephrine

In presence of TTX’

10 12 14

36 42 k 6 26 21 32 2 9

15

IS

40 41-c 31 21 335 9

16

14 52 IS 14 34 25 25 34

2

7

18

Release

in Ch’ Buffer

W)

FIG. 1. Comparison of norepinephrine release in Na + and Ch’ buffers. Each assay was seen for 20 min at 3PC. The number beside selected points is the number assigned to the chemical in Table I I but non all chemical numbers are included in the figure for the sake of clarity. The horizontal and vertical bars represent the standard deviations for the number of experiments indicated in Table 1. Compounds 20 and 22 actually left more norepinephrine in the presence of Ch + and are graphed as less than zero. The data for veratridine are represented by an open circle.

19 20 21 22 24

Veratridine

in the

release (9%released)b

In absence of TTX

Compound”

Norepinephrine

2

Norepinephrine Release by Pyrethroids Presence and Absence of TTX

Difference -4

I

3 -5

0 7

-I 6 3

2 1

II-t

I

? 7

s9+ 19 72 305

4

-7 -4

4 2

7 4 14 7 27

k 2 ? k -+

3 8 7 6 5

11 + 10 17% 72

3 0

II

1 2 I 1 3 I 2 2 I 2 2 4 3 2

u See Table I for chemical names. b All agents were tested at 30 FM final concentration. Twenty minutes were allowed for norepinephrine release. The data are expressed as averages + SD for the number of assays indicated by n. c Synaptosomes loaded with norepinephrine were added to buffer containing 3 pJ4 TTX in an ice bath and 10 min later the release reactions were started by adding the pyrethroids and transferring the tubes to a 37°C water bath.

PYRETHROIDS

191

AND RAT BRAIN SYNAFTOSOMES

well as several others. The release induced by cis-cypermethrin (22) was appreciably reversed by TTX. The minimum effective concentrations for compound 7, resmethrin (14), and tetramethrin (18) to promote norepinephrine release were in the range 3-10 FM and induction of release was concentration dependent at least up to 30 FM (Fig. 2). Testing concentrations above 30 FM were considered impractical because of the insolubility of the pyrethroids. Cn2+ Uptake and Displacement

The demonstration that rat brain synaptosomes (P2 fraction) readily took up Ca2+ in the presence of ATP but not in its absence and at each time interval tested up to 12 min resmethrin (5 @V) inhibited the uptake of Ca2+ was presented previously (6). The series of 25 pyrethroids were assessed for their effects on Ca2+ uptake at 5 FM (Table 3). The most effective inhibitors were compounds 7 and 10 and resmethrin (14), which resulted in greater than 70% inhibition. Not all compounds resulted in inhibition. Compounds 4, 12, and 13, cis-per-

-5 log c (M) FIG. 2. Concentration dependence of pyrethroids for the release of norepinephrine from rat brain P, frac,tion. The P, fraction M’as incubated with m-OPr(i) berzzyl clzrysantlzemate (7, l ), resmethrin (14, 0). and tetramethrin (18, A) for 20 min at 37°C at the concentrations indicated. Tlze vertical bars represent the starzdard deviation for n = 4 for :ero time, n = 3 for ull concentrations except the highest for whicIz n = -7.

Inhibition

TABLE 3 of Ca2+ Uptake by Pyrethroids and Related Compounds”

/

log! 100 Compound” 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24 25

% inhibition

\d

- %. inhibition

Average i SD%=

n

Obs.

40 -c 15 66 5 16 34 i 16 P 68 -c I? 68 i 14 74 5 IO 325 5 29 k 15 71 -c 17 22 t 23 P e 72 -t I2 35 2 28 e 24 + 21 21 i- IO 14-c 8 I 31 2 23 e e 3 k 16 e

4 4 4 3 4 4 5 4 8 6 2 3 2 9 6 2 3 s 3 2 2 2 2 2 2

-0.19 + 0.28 0.33 t 0.38 -0.34 t 0.39 _I 0.34 2 0.25 0.30 -c 0.28 0.47 -t 0.23 -0.34 r 0.10 PO.44 + 0.39 0.44 i- 0.39 -0.59 ? 0.34 A A 0.44 i- 0.25 PO.19 2 0.51 -J PO.65 -c 0.611 -0.62 i 0.30 1.08 k 0.73’ _I -0.42 2 0.X’ A A ~~I .49 _f

WC. 0.10 0.18 -0.17 - 1.97 0.18 0.35 0.38 -0.17 -0.40 0.35 -0.83 - 1.97 -2.31 0.30 - 0.05 0.03 - 0.62 0.28 0.36 - 2.58 0.29 -2.29

0 Tested at 5 FM. n = number of assays. Ten minutes were allowed for Ca*+ uptake for all experiments. b See Table 1 for chemical names. c The SD was determined based on the data from the number of experiments indicated by II. d The percentage inhibition from thr individual experiments was first converted to the log value and the average for the log values was determined. The percentages listed do not convert to the log values given. The calculated values were derived from Eq. [21. e The radioactivity of the test sample was higher than that of the control. J Not included in Eq. [2].

methrin (16), fenvalerate (20), cis-cypermethrin (22), MTI-800 (23), and MTI-790 (25) consistently (or for some usually) caused increases in the amount of Ca2+ incorporated. When tested at 30 PM, pyrethroids which resulted in consistent inhibition at 5 FM resulted in nearly total inhibition while others which did not inhibit at 5 FM inhibited Ca2+ uptake. Fenvalerate (20), MTI-800 (23), and MTI-790 (25) at 30 PM, however, did not inhibit Ca2+ uptake or resulted in distinctly more Ca2+ being incorporated (data not shown). Some pyrethroids displaced Ca2+ that

192

DOHERTY

was prebound to the P, material as shown in Figs. 3 and 5. In general, pyrethroids which were strong inhibitors of Ca*+ uptake were most effective in displacing bound Ca*+. Others, particularly compounds 3 and 12, cyphenothrin (19), fenvalerate (20), cis-cypermethrin (22), and MTI-800 (23), did not displace Ca*+ and up to 81% more Ca*+ was left on the P2 material than the respective control (data not shown). Lipophilicity Dependence of Norepinephrine Release and CaZf Uptake Inhibition

The log expressions for the effectiveness of the various pyrethroids to promote the release of norepinephrine [log(%release)/ (100 - %release)] and to inhibit Ca2f uptake [log(%inhibition)/( 100 - %inhibition)] were plotted (Fig. 4) versus lipophilicity (log P, where P is the n-octanollwater partition coefficient). If we consider that the percentage release or percentage inhibition caused by the pyrethroids is proportional to the amount of each compound occupying binding sites to result in stimulation of norepinephrine release or to cause inhibition of Ca*+ uptake, the log ratios as above can be regarded as binding constants. In both cases, a parabolic relationship was evident (Fig. 4) although there were some exceptions. The most notable exceptions to parabolic relationships in Fig. 4 were kadethrin (17), cyphenothrin (19), and fenvalerate (20). These agents were less effective than would be expected from their log P values as both potentiators of norepinephrine release and inhibitors of Ca*+ uptake. Ethofenprox (24) was weaker than expected as an inhibitor of Ca2f uptake but was on the parabolic curve for norepinephrine release. Conversely, tetramethrin (18) was more effective as a releaser than would be expected from its log P value but was on the parabolic curve for Ca2f uptake inhibition. Cyphenothrin (19) and fenvalerate (20) have an a-cyano group. Kadethrin (17) has a thiololactone

ET AL.

ring and ethofenprox (24) does not have an ester linkage. The structure of the alcoholic moiety of tetramethrin is considerably different from the other chemicals in this series. Thus, these compounds may not fit the parabolic curves because of significant structural differences from the other chemicals in the series. The relationships between lipophilicity and either the log expression for the releasing effects or Ca*+ uptake inhibition were assessed by regression analyses using the method of least squares. For release promoting effects an equation was formulated for 18 compounds which resulted in release and for which the log P value was available. %release 1% 100 - %release = i i -2.254 + 0.682 log P (1.092) (0.346) - O.O63(log P)’ (0.026) n = 18, r = 0.136,

[II s = 0.900;

log Popt from this equation is 5.41. Equation [2] was formulated for the 13 compounds which showed inhibitory effects on Ca*+ uptake at 5 pA4 and did not have structural exceptions: %inhibition log 100 - %inhibition

=

- 14.833 + 4.784 log P (4.473) (1.416) - 0.376(log P)* (0.110) m r = 0.926; n = 13, s = 0.175, the log Pop, for this equation equations H is the number standard deviation, and r coefficient. The numbers in

is 6.36. In both of data, s the the correlation parenthesis are

PYRETHROIDS

AND

RAT

BRAIN

SYNAPTOSOMES

193

either Eq. [I] or [2] was not significant improving the correlations.

in

DISCUSSION

Time (mln)

3. Displacement of bound Ca?+ by pyrethroids. The rat brain P, fraction preloaded with 4sCa2+ was further incubated with methanol solutions of tetramethrin (18, A), resmethrin (14, A) or MTI-790 (25, l ) at a final concentration of 30 PM and compared with samples treated with methanol only (0). The vertical bars represent the stundard deviation for n = 3. FIG.

the 95% confidence intervals. Addition of other parameter terms which represent electronic and steric factors, such as Iiammett u and van der Waals volume (VW), into

Because the most effective pyrethroids required concentrations greater than 10 FM to consistently promote release, all of the pyrethroids in the series were weak releasers of norepinephrine in synaptosomes. Based on the experiments with Ch+ and TTX, most of the pyrethroids in this series promoted norepinephrine release in )litro by a mechanism that is independent on the Na+ channel. The pyrethroids for which Na+ independent release was best demonstrated were the most effective displacers of preloaded Ca*+ (Fig. 5). Thus, elevation of intracellular Ca* + following displacement by the pyrethroids provides a reasonable alternative explanation for the transmitter releasing effects for those pyrethroids not showing Na+ dependence, because elevation of intracellular Ca*+ results in transmitter release (15). inhibition of Ca*+ uptake or blockage of the Ca*+

A

L

6

8

log P

-21

4

6

8

8

log P

FIG. 4. Relationship of the potential for pyrethroids to induce the release of norepinephrine (Part A) and to inhibit Ca2+ uptake (Part B) CO their lipophilicity (log P). The number beside each point is the number assigned CO each chemical in Table 1. The log P value for each chemical is also listed in Table 1. The vertical bars represent the standard deviation for the number of determinations made and indicated in Table 1. The data points with open circles were not included in derivation of Eqs. [I] and [2].

194

DOHERTY

Norepinephrine

Release

(%)

5. Relationship between norepinephrine release and displacement of preloaded Ca2+ by pyrethroids. The number beside each point is the number assigned to each chemical in Table I. The horizontal and vertical bars represent the standard deviations. FIG.

channel as has been demonstrated for tetramethrin (16) would not explain the increase in transmitter release induced by pyrethroids because our assays were run in Ca2+ free buffer. Pyrethroids may directly displace norepinephrine from its storage sites to give the appearance of promotion of release but proof of this is beyond the scope of this paper. The effectiveness of most of the pyrethroids in this series both in promotion of norepinephrine release and in inhibition of Ca2+ uptake depended mostly on lipophilicity (log P). The log Port for norepinephrine releasing effects (5.41) is reasonably close to the log P,,, for Ca2+ uptake inhibition (6.36) considering that there is a sixfold difference in the concentrations at which Ca2+ uptake inhibition (5 PM) and norepinephrine release (30 p&I) experiments were assessed. A log PO,, of 6.62 was found for this series of pyrethroids when Ca2+ uptake inhibition data from crayfish axon homogenates were analyzed by regression analysis (17). Thus, the same physical laws apparently govern the interaction of noncyano pyrethroids with nerve membrane with respect to Ca2+ uptake inhibition. The compounds must penetrate through a number of barriers (18) to arrive at site(s) where mechanisms controlling ATP-dependent Ca2+ uptake and Ca2+ storage are located and the penetration rate appears to

ET AL.

be governed by lipophilicity. The Na+ independent norepinephrine release thus shows the same parabolic relationship because it results from the effects of the pyrethroids on Ca2+. Simple penetration phenomena, however, do not explain why some pyrethroids showed Na+ dependent norepinephrine release and others consistently showed increases in Ca2+ bound to the synaptosomes. Although the pyrethroid effects on Ca*+ may explain the release promoting properties of the noncyano pyrethroids in vitro, there is no basis for correlating the effects of the pyrethroids on Ca*+ with known lethality in vivo. The toxicity (LD,,) of various pyrethroids to rats and mice following intracerebroventricular injection has been reported by other workers (19-21). The pyrethroids which were most toxic when injected intraventricularly were among the weakest Ca2+ uptake inhibitors. For example, cis-cypermethrin, fenvalerate, and fenpropathrin (LD,,s = 0.6, 1.O, and 6.1 p,g/g of brain, respectively, (19)) did not inhibit Ca2+ uptake. One of the most effective Ca*+ uptake inhibitors, trans-resmethrin, and the moderate Ca2+ inhibitors, trans-permethrin and trans-tetramethrin, had intraventricular LD,,s of >860 pg/g of brain (19). Direct involvement of noncyano pyrethroid effects on the Na+ channel in synaptosomes in vivo could still be related to toxicity, however, if our in vitro assay greatly underestimates the interaction of pyrethroids with the Na+ channel in riro. We recognize that in invertebrate axons only as little as I% or less of the Na+ channels need to be modified by the pyrethroids (22) to produce repetitive discharges. Activation of about 1% or less of the Na+ channels in synaptosomes may possibly result in some physiological/behavioral responses but may be insufficient to promote detectable transmitter release. Transmitter receptor/ionophore complexes such as for GABA (23), glutamic acid (24), or ACh (25) have been suggested as target sites for pyrethroid action. The

PYRETHROIDS

AND RAT BRAIN SYNAPTOSOMES

concentrations of pyrethroids necessary to affect these systems in vitro are the same or higher than the concentrations for the most effective pyrethroids to inhibit Ca*+ uptake. It is still possible that the effects of some pyrethroids on Ca*+ metabolism may contribute to the overall neuro- and general biochemical pharmacology of these agents. In summary, the effects of many chemicals in this series of pyrethroids on Ca*+ uptake inhibition and norepinephrine release were shown to vary mostly with lipophilicity but not with a specific property of this class of chemicals. With the exception of some of the a-cyan0 analogs, this series of pyrethroids did not display a veratridine-like effect on the Na+ channel in rat brain synaptosomes. Promotion of transmitter release in rat brain in vitro by most of these pyrethroids correlated better with their effects on Ca*+ metabolism rather than on the Na+ channel. Ca*+ metabolism effects of the pyrethroids in vitro do not directly correlate with known in vim toxicity. ACKNOWLEDGMENTS The authors thank Roussel UCLAF, Sumitomo Chemical Co., and Mitsui Toatsu Chemical Co. for supplying pyrethroid samples. The calculations were performed with a FACOM M-382 computer at the Data Processing Center of this university. J.D.D. greatly appreciates the fellowship provided by the Japan Society for the Promotion of Science and the U.S. Environmental Protection Agency for granting a temporary leave of absence which enabled him to participate in this project. REFERENCES 1. T. Narahashi, Effects of insecticides on nervous conduction and synaptic transmission, in “Insecticide Biochemistry and Physiology,” (C. F. Wilkinson, Ed.), p. 327. Plenum, New York/ London, 1976. 2. J. E. Casida, D. W. Gammon, A. H. Glickman. and L. J. Lawrence, Mechanisms of selective toxicity of pyrethroid insecticides, Annu. Rev. Pharmaco/. Toxicol. 23, 413 (1983). 3. M. Ohta, T. Narahashi, and R. F. Keeler. Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons. J. Pharmacol. Exp. Ther. 184, 134 (1973).

195

4. J. Cunningham and M. J. Neal, On the mechanism by which veratridine causes a calcium independent release of y-aminobutyric acid from brain slices, Brif. J. Pharmacol. 73, 655 (1981). 5. J. P. Abita, R. Chicheportiche. H. Schweitz, and M. Lazdunski. Effects of neurotoxins (veratridine, sea anemone toxin, tetrodotoxin) on transmitter accumulation and release by nerve terminals, Biochemistry 16, 1838 (1977). 6. J. D. Doherty, C. J. Lauter.and N. Salem, Jr., Synaptic effects of the synthetic pyrethroid resmethrin in vivo. Camp. Biochem. Physiol. C 84, 373 (1986). See also Pharmacologist 25, 23 1 (Abstract 673) (1983). 7. R. A. Nicholson, R. G. Wilson, C. Potter. and M. H. Black. Pyrethroid- and DDT-evoked release of GABA from the nervous system in vitro, in “Pesticide Chemistry, Human Welfare and the Environment,” Vol. 3, “Mode of Action, Metabolism. Toxicology,” (J. Miyamoto and P. C. Kearney, Eds.), p. 751, Pergamon. New York, 1983. 8. 0. H. Lowry, N. J. Rosebrough. A. L. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Rio/. Chem. 193, 265 (1951). 9. M. P. Blaustein. E. M. Johnson, Jr., and P. Needleman, Calcium dependent norepinephrine release from presynaptic nerve endings in vitro, Proc. Natl. Acad. Sri. USA 69, 2237 (1972). 10. F. Guerrero-Munoz. K. V. Cerrata, M. L. Guerrero, and E. L. Way, Effect of morphine on synaptosomal Ca*+ uptake. J. Pharmacol. Exp. Ther. 209, 132 (1979). 11. S. Nakagawa, N. Okajima, T. Kitahaba. K. Nishimura, T. Fujita, and M. Nakajima, Quantitative structure-activity studies of the substituted benzyl chrysanthemates: 1. Correlation between symptomatic and neurophysiological activities against American cockroaches, Pestic, Biochem. Physiol. 17, 243 (1982). 12. K. Nishimura. T. Kobayashi, and T. Fujita, Symptomatic and neurophysiological activities of new synthetic non-ester pyrethroids. ethofenprox, MTI-800, and related compounds, Pestic. Biochem. Physiol. 25, 387 (1986). 13. M. Omatsu, K. Nishimura, and T. Fujita, Quantitative structure-activity studies of substituted benzyl chrysanthemates: 8. Physicochemical properties and neurophysiological effects on membrane potentials of crayfish giant axon, Pestic. Biochem. Physiol. 24, 192 (1985). 14. C. Hansch and A. J. Leo. “Substituent Constants for Correlation Analysis in Chemistry and Biology,” p. 13, Wiley, New York, 1979. 15. W. W. Douglas, Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. Brit. J. Pharmacol. Chemother. 34, 451 (1968).

DOHERTY

196

16. A. Tsunoo, M. Yoshii, and T. Narahashi, Differential block of two types of calcium channels in neuroblastoma cells, Biophys. J. 47, 433a (1985). 17. J. D. Doherty, K. Nishimura, N. Kurihara. and T. Fujita, Quantitative structure-activity studies of the substituted benzyl chrysanthemates: 9. Calcium uptake inhibition in crayfish nerve cord and lobster axon homogenates in virro by synthetic pyrethroids, Pesfic. Biochem. Physiol.

ET AL.

21. 22.

23.

25, 295 (1986).

18. J. T. Penniston, D. L. Bentley, and C. Hansch, Passive permeation of organic compounds through biological tissues: A non-steady-state theory, Mol. Pharmacol. 5, 333 (1969). 19. L. J. Lawrence and J. E. Casida, Pyrethroid toxicity: Mouse intracerebral structure-activity relationships, Pestic. Biochem. Physiol. 18, 9 (1982). 20. C. G. Staatz. A. S. Bloom, and J. J. Lech. A pharmacological study of pyrethroid neurotox-

24.

icity in mice. Pestic. Biochem. Physiol. 17, 287 (1982). A. J. Gray and J. Rickard, Toxicity of pyrethroids to rats after direct injection into the central nervous system. Neurotoxicology 3, 25 (1982). A. E. Lund and T. Narahashi, Dose dependent interaction of the pyrethroid isomers with sodium channels of squid axon membranes, Neurotoxicology 3, 11 (1982). L. J. Lawrence and J. E. Casida, Stereospecitic action of pyrethroid insecticides on the y-aminobutyric acid receptor-ionophore complex. Science 221, 1399 (1983). G. G. Staatz. A. S. Bloom, and J. J. Lech. Effects of pyrethroids on [3H]-kainic acid binding to mouse forebrain membranes, Toxicol. Appl. Pharmacol.

64, 566 (1982).

25. M. A. Abbassy, M. E. Eldefrawi, and A. T. Eldefrawi. Pyrethroid action on the nicotinic acetylcholine receptor/channel. Pestic. Biochem. Physiol. 19, 299 (1983).