EFFECT OF ORGANOPHOSPHORUS COMPOUNDS ON ACETYLCHOLINE SYNTHESIS IN BRAIN

Japan. J. Pharmacol. 26, 249-254 (1976)

EFFECT

OF

249

ORGANOPHOSPHORUS

ACETYLCHOLINE Makoto

COMPOUNDS

SYNTHESIS

MURAMATSU

IN

ON

BRAIN

and Kinya KURIYAMA

Department of Pharmacology, Kyoto Prefectural University of Medicine, Kamikyo-kii Kvoto 602. Japan Accepted January

12, 1976

Abstract-Effects of parathion and di-isopropyl fluorophosphate (DFP) on acetyl choline (ACh) synthesis in the mouse brain were investigated. In addition to well known cholinesterase (ChE) inhibition, parathion showed inhibitory effects on the activity of synaptosomal choline acetyltransferase (ChAc), and on the uptake of [14C methyl]-choline and ACh synthesis in subcellular fractions of the brain. DFP inhibited ChE activity, but had no significant effects on the choline uptake and ACh synthesis per se. Possible significance of these findings in the pharmacological actions of organo phosphorus compounds is briefly discussed. It has been well established effect on the activity compounds

that

organophosphorus

of cholinesterase

(ChE),

may not alter the turnover

but other pharmacological

organophosphorus

that

compounds,

In the present

diethyl-3,

actions

of these

decreases

on the activity

in subcellular

fractions

MATERIALS

ACh synthesis

of choline

mitochondria)

Synaptosomal

the procedure

type B).

of Gray and Whittaker

assay procedure

of Fonnum

studies, choline concentrations For assay blanks, were ruptured

and DFP

hypotonically

as an enzyme preparation.

nuclear

on ChE activity, (ChAc)

but

and on the

of subcellular

fractions,

in a glass homogenizer

(P,) fraction

by centrifuging

(P3) fractions

at 1,000

g for

at 12,000 h g for

were obtained

according

to

(3). ChAc activity was determined

(4) using []-14C]-acetyl

according

from the reaction

by suspending

to the micro

CoA (S.A.: 3.7 mC/m mol).

were varied for the range of concentrations

choline was omitted

one of the

brain were examined.

For the separation

was obtained

and microsomal

Enzymatic and chemical assays:

Kobayashi

AND METHODS

After separating

(P.,) fraction

(P,-B)

ChE.

(DDVP),

in the rat brain in vitro.

of whole brain with 0.32 M sucrose was prepared

with Teflon pestle (Thomas 10 min, crude

phosphate

acetyltransferase

from the mouse

Male ddy mice weighing 20-30 g were used. a 10°o homogenate

used at high concentrations

(ACh) solely by inhibiting

2-dichlorovinyl

study, not only the effect of parathion

also effects of these drugs of choline

that ChE inhibitors

rate of acetylcholine

et al. (2) have also reported

30 min.

have an inhibitory

remain unclear.

Hanin et al. (1) suggested the possibility

uptake

compounds

mixture.

into 5 mM phosphate

For kinetic

of 0.085-10

Synaptosomal

mM.

fractions

buffer (pH 6.8) and used

250

11. MURAAVATSU Acetylcholinesterase

(AChE)

activity

et al. (5). Lactic dehydrogenase were determined

respectively

al. (7).

content

Protein

Determination fraction

(protein

(final concentration)

10.000

and

Following

pellet

Radioactivities

using a Packard

was centrifuged

was transferred

was transferred

scintillation

of P2 fraction

and [14C-methyl]-ACh

at 1,000 ;< g for 5 min and the supernatant supernatant

of P_ fraction

methanol

suspension

ACh,

and

procedure

were directly

of the extract

applied respectively

[14C-methyl]-choline described

iodoplatinate

by Marchbanks

of 4.5 / (W/V) KI in ethanol-water off from the plate and dispersed

the mixture

including

Model

applied

(1 :1000 (V/V)).

thion and DFP were added into various

mixture

these procedures and 0.5 ml of me

obtained

after the centri

acid-water by spraying

were

choline and

according

to the

mixture (8:2:1 :3) the dry plate with

acid was added to 20 parts

Each of the identified

vial containing

Ten %d of fractions

with authentic

were separated

(V/V)) chloroplatinic

was measured

were

in both supernatant

and the supernatant

(9). Butanol-ethanol-acetic

uptake were carried out at pH 6.8 to prevent

the reaction

at 25'C

fractions

plate together

spots was scraped

I ml of 0.1 N NH3 in ethanol.

(1:1 (V/V)) was added to decolourize

as described

the measurements

solubilizing

fractions

to a thin layer chromatography.

[«C-methyl]-ACh

in a counting

vial by de

3390.

After repeating

to a near dryness

Na2S2O3 in ethanol-water

and the radioactivity

All experiments

was collected.

the supernatant

reagent (One part of aq. 5

pellet

at

ice for 30 min, each tube was centrifuged

Choline and ACh spots were identified

One tenth ml of 5°i(, WV)

mixture centrifuge

in 0.5 ml of I N formic acid-acetone

in crushed

from pellet fractions

and

vial after

solubilized

was also determined

to thin layer chromatographic

was used for elution.

10 ,eM

centrifuged

directly to a counting

with [14C-methyl]-choline,

was brought

On the other hand,

fugation

0.1 mM

the incubation,

to a counting and

The P.2 pellets were suspended

thanol was added.

(P2)

adding

and immediately

spectrometer

synthesized

mixture (15:85 (V/V)) (8). After standing

twice, the combined

After

mol), the reaction

Following

to each tube,

in the supernatant

Tri-carb

the incubation

and pellet fractions.

mitochondrial

(pH 6.8) containing

was used.

54.9 mC/m

for 15 min.

was added

The supernatant

the remaining

with hyarnine.

The crude

solution

mg/mI)

(S.A.:

activities

were chilled in ice and I ml of ice cold 0.32 M sucrose containing

choline

g for 10 min.

measured

2.3-5.7

of Ellman

(MDH)

of Lowry et al. (10).

and ACh synthesis:

content:

to the method

of Bergmeyer et al . (6) and Mehler et

by the method

of ['4C-methyl]-choline

of unlabelled

cantation

to the method

at 0°C and 37"C respectively

tubes used for the reaction 1.0 mM

according

according

and malic dehydrogenase

in 1 ml of the Krebs-bicarbonate

sulfate

was incubated

was assayed activity

was determined

of' Choline uptake

suspended

physostigmine

(LDH)

& K. KURIYAMA

of enzyme

the decomposition

previously. activities

as well as choline

of added parathion.

test systems after suspending

Para

with propyleneglycol.

RESULTS EJJeet of parathion In the brain significant

and DIP

on ChAr « etirities

homogenate

and synaptosomal

effect on ChAc activity,

whereas parathion

fractions

UFP

(0.01-10

niM)

lead no

(1-10 mM) showed a dose-dependent

ORGANOPHOSPHORUS

Flu.

1.

COMPOUNDS

Effect of parathion p ;0.05,

ON

ACh

and DEP on ChAc activity

Compared

with control

SYNTHESIS

251

in brain homogenate

value.

Fic. 2. Effect of parathion on Lineweaver-Bulk plots of ChAc activity in brain homogenate and synaptosomal fraction. homogenate: Km =1.43 mM, synaptosomal : Km 1.81 mM

inhibition

on ChAc activity

parathion

was non-competitive.

In preparations

(Figs.

I and 2).

of homogenate,

microsomal

AChE activity was found in microsomal aptosomal

fraction.

In these fractions The inhibitory (12).

These results are essentially the 0.05 mM of parathion

parathion

and synaptosomal

L.Ji ct of parathion

fractions

effect of

the highest

and the lowest activity was found in syn in good parallel with previous reports (l l ).

and DFP inhibited

1 mM had no significant

in the brain homogenate.

parathion on ChAc activity on general enzyme proteins.

DFP

fraction

in Fig. 2, this inhibitory

effect of DFP on AChE activity is reportedly

In addition,

LDH assayed

As shown

These results

significantly

stronger

AChE activity.

than that of parathion

effect on the activities

of MDH

suggest that the inhibitory

may not be a simple reflection

and

effect of

of the effect of this compound

on [14('-na t/u 1]-cholinc uptake

(4 mM) showed a slight inhibitory

effect on ['1C-methyl]-choline

uptake

into P,

252

M.

fraction,

but this inhibitory

statistically

significant.

hand, parathion cantly

trations

uptake

the

(Table

of [14C-methyl]-choline of 2.5-25 ,N,

apparently

parathion

competitive

& K. KURIYAMA

effect was not On

(4 mM) inhibited

the choline

the range

MURAIVIATSU

inhibitory

other signifi 1).

At

concen showed effects

on the choline uptake (Fig. 3). As shown

in Table 2, the inhibitory

effect of parathion uptake

was detected

observed

at 0`'C.

that parathion

(1 m M) on the choline at 37 C, but was not These

results

FIG. 3. Effect of parathion on ['4C-methyl] choline uptake into crude mitochondrial fraction. K m -.26.8 It M,

Ki

42.7 /LM

indicate

has inhibitory effects on temperature and possibly energy dependent pro

cesses of the choline uptake. Effect of parathion on ACh synthesis Following the incubation at 37°C for 15 min of P, fraction with [14C-methyl]-choline, both [14C-methyl]-choline and ['4C-methyl]-ACh extracted from the incubation

mixture

TABLEI. Effect of organophosphorus compounds on ['4C-methyl]-cholineuptake into crude mitochondrial (P2) fraction of mouse brain

The meanS.E.M. obtained from four separate experiments. The incubation medium contained 10 n mol;'ml of ['4C-methyl]-choline and 2.2-5.9 mg protein of P2 fraction. p ;0.05, Compared with control value. TABLE2. Effect of parathion on uptake of ['4C-methyl]-choline by crude mitochondrial (P2) fraction at 0 C and 37 C

The mean _ S.I.M. obtained frmm three separate experiments. The incubation ittedium contained l() n mol/ml of ['4C-methyl]-choline mCijm mol) and 2.7 3.4 mg protein of P2 fraction.

(S.A.: 54.9

ORGANOPHOSPHORUS TABLE3. Effect of parathion fraction at 37-C

COMPOUNDS

ON ACh SYNTHESIS

on acetylcholine synthesis in crude mitochondrial

253 (P2)

The mean-S.E.M. obtained from four separate experiments. The incubation medium contained 10 n mol/ml of [14C-methyl]-choline(S.A.: 54.9 mCi/m mol) and 2.3-3.7 mg protein of P2 fraction. * p<0 .05, ** p<0.02, Compared with each control. were analyzed by a thin layer chromatographic

procedure.

More than 90% of the radioactivity was recovered as [14C-methyl]-choline and [14C methyl]-ACh. In pellet fractions, the conversion rate (expressed as ACh x 100/choline+ACh)

for

control was 24.2, whereas rates for 1 mM and 4 mM of parathion were 18.1 and 16.2, re spectively (Table 3). These results clearly indicate that parathion has inhibitory effects on ACh synthesis. In supernatant fractions, the conversion rate for control was 3.4 and showed a tendency to decrease with addition of parathion, but these effects were not statistically significant. DISCUSSION One of the most interesting findings in this study is that parathion, which has been classified as one of ChE inhibitors and known as an organophosphorus

compound, also

has inhibitory effects on the activity of ChAc and on the uptake of choline into brain sub cellular fractions. The extent of inhibitory effects of parathion on the ACh synthesis in the brain tissues was larger than the inhibitory effects on ChAc activity or on the choline uptake, suggesting that the inhibition of ACh synthesis by parathion may be a reflection of the inhibitory effects of this compound

on both parameters.

Hanin et al. (1) suggested the

possibility that cholinesterase inhibitors used at high concentrations do not affect the steady state of ACh solely by inhibiting ChE, but inhibitory effects of these compounds on choline uptake might also be involved as one of the main control mechanisms. On the other hand, Kobayashi et al. (2) reported that DDVP, an anti-AChE agent, diminished ACh synthesis by inhibiting ChAc activity.

Our results indicate that parathion has inhibitory effects on

both ChAc activity and choline uptake, simultaneously. Parathion and DFP have long been classified in the same category as organophosphorus ChE inhibitors.

These compounds,

however, have a different effect on ACh synthesis;

254

M. MURAMATSU

Parathion

inhibits ACh synthesis

It is well documented (15), possibly

synthesis, inhibits

in the brain tissue, while DFP has no such a property.

that neurotoxic

effects of parathion

due to the weaker inhibitory

than the latter.

However,

another

& K. KURI YAMA

if we consider differential

possible factor

both ChE and ACh synthesis,

than DFP.

effects on ACh synthesis Although of ChAc certain,

the significance and choline

of inhibitory

uptake

also the inhibitory

effects of parathion

and thus the increase

on ChE activity and DFP on ACh

of parathion

required

higher than that required effects of parathion

in terms of pharmacologic

Parathion

of ACh in neuronal

toxicity of these compounds,

The concentration

is undoubtedly

it must be emphasized

lower than DFP

compound

may also be added to explain these differences;

the agent which plays a main role in neuronal case of parathion

are generally

effect of the former

tissues,

is lower in the

to exhibit inhibitory

for the inhibition

of ChE.

on ACh synthesis via inhibitions actions

that not only the well known

effects on ACh synthesis must be considered

of this compounds

inhibition

is un

of ChE activity,

but

when a high dose of parathion

is used. Recently

stryptoridine

These compounds a hypothesis

analogues

also inhibit

have been introduced

ChE activity

that both esterase and acetylase

these agents (14). Similar mechanisms

at relatively

as a potent

ChAc inhibitor

high concentrations,

have similar functional

(13).

and there is

groups to combine with

may be involved in the inhibitory

effects of parathion

on both ChE and ChAc activities. Acknowledgement: 1974) from the Ministry

This work was supported of Education,

in part by research

grant (No. 987015,

Japan.

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