Influence of exercise and ethanol on cholinesterase activity and lipid peroxidation in blood and brain regions of rat

Reg.

ELSEVIER

NeumPsyc~pphnrmacd.

& Bti. Psyhiat. 1997, Vol. 2 1, pp. 059-670 Copyright 0 1997 Elsevier Sdence Inc. Printed in the USA. All rights reserved 0276-5646/97 $32.00 + .oO

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INFLUENCE OF EXERCISE AND ETHANOL ON CHOLINESTERASE ACTIVITY AND LIPID PEROXIDATION BLOOD AND B&UN REGIONS OF RAT

IN

KAZIM HUSAIN and SATU M. SOMANI Southern Illinois University School of Medicine Department of Pharmacology Springfield, IL, USA (Final form - January, 1997)

Abstract Husain, Kazim and Satu M. Somani: Influence of exercise and ethanol on cholinesterase activity and lipid peroxidation in blood and brain regions of rat. Prog. Neuro.-Psychopharmacol. & Biol. Psychiat. 1997,a: pp. 659-670. 0 1997 Elsevier Science Inc. 1. This study elucidates the interaction of acute exercise and single ethanol intake on cholinergic enzyme and its relationship to lipid peroxidation in the blood and brain regions of the rat. 2. Butyrylcholinesterase (BuChE) in plasma and acetylcholinesterase (AChE) in brain regions as well as lipid peroxidation (MDA) were assayed in 1) sedentary control rats; 2) after acute exercise (100% VO1,,,); 3) ethanol 20% (1.6 gm/kg, p.0.); 4) exercise and then ethanol 20% (1.6 gm/kg, p.0.). 3. Acute exercise significantly increased BuChE activity (155% of control) in plasma and decreased AChE activity (60% of control) in the corpus striatum with a significant increase in the striatal MDA level (254% of control). Ethanol significantly decreased AChE activity only in striatum (86% of control) with a significant increase in striatal MDA level (132% of control). 4. The combination of exercise and ethanol 20% (1.6gm/kg, p.0.) significantly increased BuChE activity (123% of control) in plasma, and decreased AChE activity (76% of control) in striatum with significant increase in striatal MDA level (147% of control). 5. Acute exercise, single ethanol 20% (1.6 grn/kg, p.o.) intake and the combination selectively inhibited striatal AChE, and the inhibition was correlated with increased lipid peroxidation indicating perturbation of motor function. The combination reduced the peripheral stress response caused by exhaustive exercise. Keywords: acetylcholinesterase. -2: cholinesterase

brain regions, butyrylcholinesterase,

acetylcholinesterase (AChE), acute exercise (ChE), ethanol (Et), malondialdehyde (MDA)

ethanol, exercise, lipid peroxidation

(AE),

butyrylcholinesterase

(BuChE),

Introduction Earlier studies reported that physical exercise enhances the inhibition of cholinergic by cholinesterase

inhibitor (physostigmine)

enzymes elicited

in cerebral and peripheral tissues of the rat (Dube et al, 1990;

Dube et al 1993; Somani et al, 1991). Exercise is known to cause oxidative stress on the nervous syste659

660

K. Husam and S.M. Somani

and increase the lipid peroxidation neuronal

membrane

permeability.

(Somani, 1994; Somani et al, 1996). which may lead to alteration in Similarly,

ethanol has also been reported to be stressful

(Tabakoff et al, 1978) and exerts an oxidative Nordmann.

1987).

Ethanol after ingestion

stress response

to mice

on the nervous system (Bondy,

is quickly absorbed

from the gastrointestinal

1992;

lumen into

circulation and can readily cross the blood brain barrier entering the areas of the brain (Ritchie, 1985). Due to high lipid solubility, ethanol also changes the permeability and fluidity of the synaptic membrane as well as the activities of membrane bound enzymes (Collins et al, 1984; Lasner et al. 1995; Sun and Smorajski. 1970).

Acetylcholinesterase

is a membrane bound enzyme with lipid dependence (Lasner et al. 1995: Ott, 1985).

and its distribution is different in specific brain regions (Appleyard et al. 1986: Husain and Vi.jayaraghavan, 1989; Somani et al, 1991); ethanol may likely influence the enzyme activity in a differential

pattern.

brain utilizes 20% of the oxygen consumed by the body, contains high levels ofperoxidizable low levels of antioxidants. hypothesis

It may actually be more vulnerable to peroxidative

is that acute exercise, single ethanol intake, and the combination

peroxidation

of neural membranes

damage.

The

lipids and

Therefore. the

are likely to influence lipid

and of the membrane bound acetylcholinesterase

enzyme in specific

brain regions. Each region of the brain has different responses to exercise (Somani et al. 199 1; Somani et al, 1995) or ethanol (Rawat. 1976; Somani et al. 1996) because each region has a separate level of peroxidizable

unsaturated lipids (Nordmann,

enzymes (Appleyard

1987; Somani et al., 1996: Uysal et al, 1989). cholinergic

et al, 1986; Somani et al, 1991). and cholinergic

innervation

(Arneric et al.. 1990:

Eckstein et al, 1988; Matin and Husain, 1985). Thus each brain region may receive a different amount of stress.

There is a paucity of information

concerning

the interaction

of exercise

cholinergic system. Only a few reports have demonstrated the influence ofethanol

and ethanol on the

on exercise performance

in human (Blomquist et al, 1970; Houmard et al. 1987; Kendrick et al. 1993: Side11 and Pless. 1971). Since exercise

and ethanol have been known to cause an oxidative

cholinergic system (Bondy, 1992: Hashem-Zadeh-Gargari

stress response

and interact with the

and Mandel. 1989; Somani et al, 199 1; Somani,

1994; Somani et al. 1995), it is essential to study the interactive effects of both on membrane-bound and lipid peroxidation

in brain regions of rat. Therefore, the specific aims of this study were:

determine whether acute exercise. single ethanol intake. and the combination influence

the cholinesterase

activity in the blood and sub-regions

correlation between cholinesterase rat.

AChE 1) to

of these two stressors would

of the brain; and 2) to establish

enzyme activity and lipid peroxidation

a

in specific brain regions of the

Exercise, ethanol/cholinesterase

661

Methods

Acetylthiocholine

iodide. butyrylthiocholine

iodide and 5’,5’ - dithiobisnitrobenzoic

acid were obtained

from the Sigma Chemical Co. (St. Louis, MO). Coomassie protein assay reagent was purchased from the Pierce Co. (Rockford, IL). _Animals Adult male Fisher-344 rats weighing 205-230 gm from Harlan Industries (Indianapolis, in this study. The rats were acclimatized for 5 days to the facility prior to starting

IN) were used

the experiments.

were fed ad libitum with Rodent Laboratory Chow (Ralston Purina Company, Indianapolis,

Rats

IN) and tap

water. Rats were randomly divided into four groups and treated as follows:

I.

Sedentarv Control (SC): of inclination minutes

Six rats were put on the treadmill belt at a speed of 2 m/min and 0”angle

in the Omnipacer

for equivalent

treadmill (Omnitech

handling.

Electronics,

Inc., Columbus,

OH) for five

They received equivalent volume of normal saline orally via

orogastric tube. II.

Acute Exercise (AE): Four rats were acutely exercised on the treadmill at 100% VOZmax.The speed of the belt and angle of inclination were increased gradually from 8.2 m/min to 30.3 m/min and from 0” to 12.5”, respectively,

as described earlier (Dube et al, 1990; Dube et al, 1993; Somani et

al, 1991). The oxygen consumption

and heat production

Oxyscan System (Omnitech Electronics,

III. Ethanol (Et):

in individual rats were recorded by the

Inc.).

Four rats were given ethanol 20% (v/v) at a dose of (1.6 gm/kg, p.o.) via orogastric

tube. IV. Acute Exercise and Ethanol (AE + Et): Six rats were exercised on the treadmill as described in group II and 5 min after exercise, animals were given ethanol as described in group III . Rats in all the groups were sacrificed 30 min post treatment by decapitation

between 9:00-l 1:OOa.m.

to minimize the Circadian cycle effects. Preparation of Tissue Extract: Blood was collected in heparinized vials and centrifuged at 1000 rpm for 10 min to separate the plasma. cerebellum, immersed

Heads were collected in ice water and brain regions - cerebral cortex,

medulla, corpus striatum and hypothalamus in liquid nitrogen and stored at -80°C.

were isolated.

Brain regions were immediately

Brain regions were homogenized

phosphate buffer (pH 7.0) containing 0.1 mM EDTA to give 5% homogenate

(“‘/J.

in cold 50 mM

K. Husain and S.M. Somani

662

Enzvme Assays Acetylcholinesterase

(EC 3.1.1.7): Its activity was determined

method of Ellman et al. (1961).

In a cuvette.

in brain regions according to modified

100 ul of tissue extract was added to 780 Pl of 0.1 M

phosphate buffer pH 8. One hundred ~1 of 0.01 M dithiobisnitrobenzoic

acid (in 0.1 M phosphate buffer

pH 7.0) was added to the cuvette. The reaction was started after the addition of 20 pl of acetylthiocholine as a substrate, and optical density was recorded at 4 I2 nm every 30 seconds up to 4 minutes using a Hitachi U-2000

spectrophotometer.

computerized

The

program.

hydrolyzed/min/mg

The change in optical density enzyme

activity

was

and enzyme

expressed

activity was calculated

as umoles

by

of acetylthiocholine

protein.

Butyrylcholinesterase

(,EC 3. I. I .8): Its activity was determined

in plasma by the modified method of

Ellman et al. (1961). In a cuvette containing 920 ul of 0.1 M phosphate buffer (pH 8), 10 ~1 of plasma and 50 u1 of 0.01 M dithiobisnitrobenzoic started with the addition.of

acid (in 0.1 M phosphate buffer pH 7) was added. The reaction was

20 ~1 of 0.075 M butyrylthiocholine

as substrate and optical density was

recorded at 4 I2 mn every 30 set up to 4 minutes. The change in optical density per minute and the enzyme activity was calculated by a computerized butyrylthiocholine

hydrolyzediminlmg

Linid Peroxidation

Assav

program.

The specific activity was expressed

as pmoles of

protein.

This assay is used to determine malondialdehyde

(MDA) level as described by Ohkawa et al. (1979).

Two hundred ul of tissue homogenate was added to 50 ~1 of8.1% sodium dodecyl sulfate (SDS), vortexed, and incubated for IO minutes at room temperature. 3.5) and 375 pl ofthiobarbituric

Three hundred seventy five pl of 20% acetic acid (pH

acid (0.6%) were added and placed in a boiling water bath for 60 min. The

samples were allowed to cool at room temperature, vortexed and centrifuged at 1000 RPM for 5 minutes.

then 1.25ml of butanol:

pyridine (15: 1) was added.

Five hundred u1 of the colored layer was measured

at 532 nm using 1.1.3.3-tetraethoxypropane as a standard.

Protein Assay Protein in plasma and tissue homogenate was determined by the method of Read and Northcole (1981) using bovine serum albumin as standard.

Data Analvsis Data were expressed as mean i S.E.M. and analyzed using one way analysis of variance (ANOVA) and group comparisons

by Tukey’s studentized

Range Test at significant level 0.05.

Exercise, ethanol/cholmesterase

663

Results The effect of acute exercise (AE), ethanol (Et) and the combination cholinesterase

activity

butyrylcholinesterase

of the rat is depicted

in Table

1.

of both (AE + Et) on plasma

AE significantly

increased

plasma

(BuChE) activity (155% of control) (F = 3 1.5, p
plasma BuChE activity.

The combination

of AE and Et significantly

increased plasma BuChE activity

(123% of control) (F = 15.4, pcO.05). Table 2 shows the effect of AE. Et and the combination (AE + Et) on AChE activity in brain regions of the rat. AE significantly

decreased

AChE activity in the corpus striatum (60% of control) (F = 25.8,

p
The combination

of AE and Et resulted in a significant decrease in AChE activity

in the corpus striatum (76% of control) (F = 21.5, p~O.001) whereas enzyme activity did not significantly change in other brain regions (Table 4).

Table 1 Effect of Acute Exercise (100% VOZmax)and Ethanol (1.6 gmkg, p.o.) Ingestion on Butyrylcholinesterase Activity in the Plasma of Rats.

Groups

Butyrylcholinesterase Activity * (Piasma)

Percent Increase

1. Sedentarv Control (n=6)

3.88 * 0.17

-___

2. Acute Exercise (n=4)

6.01 * 0.30a

55

3. Ethanol (n=4)

4.06 f 0.18

5

4.78 f 0.37b’C

23

4. Acute Exercise + Ethanol (n=S)

Data are mean + SEM; *b.tmoles butyrylthiocholine hydrolyzed/min/mg protein; a - significantly different from group 1 (p < 0.01); - significantly different from group 1 (p < 0.05); ’ - significantly different from group 2 (p < 0.05)

664

K. Husam and SM. Somani Table 2 Effect of Acute Exercise ( 100% VOrma,) and Ethanol (1.6 gm/kg, p.o.) Ingestion on Acetylcholinesterase Activity in Brain Regions of Rats, Brain Regions (acetylcholinesterase Cerebral Cortex

Groups

Corpus Striatum

Medulla

activity)*

Cerebellum

Hypothalamus

1. Sedentary Control (n=6)

18.20i I.55

68.83t 2.68

49.69+ 4.07

21.62+ 1.79

43.78* I.75

2. Acute Exercise (n=4)

15.12* 0.75 (-17%)

41.3I"i 3.21 (-40%)

38.41-t3 71 (-23%)

22.99+ 3.13 (+6Oa)

41.97 Ik1.50 (-4%)

3. Ethanol (n=4)

22.62+ 1.69 (+24%)

59.00bgd i 2.24 (-14%)

59.51'* 7.79 (+20%)

19.80+ 1.68 (-8%)

49.42i 3.48 (+13%)

4. Acute Exercise + Ethanol (n=5)

?3.01'+ 1.69 (+26%)

52.42"e'f+1.37 54.31f 7.12 (-2496) (+9%)

21.64i 1.23 (0%)

48.08t4.59 (+lo%)

Data are mean * sem; * bumoles Acetylthiocholine hydrolyzediminimg protein: ’ - significant (p i 0.01) ‘.; - significant (p < 0.02) compared to group 1; ’ - significant (p < 0.01) compared 1 - srgnn‘icant (p i 0.01) compared with group 2; e - significant (p < 0.02) compared with group 2; ’ - significant (p < 0.05) compared with group 3: g - significant (p < 0.05) compared with group 2; Values in parentheses indicate percent change (+) increase and (-) decrease ;;Tgy;pw;,hpup

The changes in lipid peroxidation (MDA) due to AE, Et, and AE + Et in plasma and brain regions of the rat is presented

in Table 3. No significant

exercise or the combination

ofAE

change in the plasma MDA level was observed after acute

and Et. However, the MDA level decreased slightly (79% of control)

in the plasma after Et treatment. AE significantly elevated MDA levels in corpus striatum (254% of control) (F = 46.87, p
(F = 13.5, p
increased the MDA level in the corpus Striatum (132% of control) (F = 15.2. p
MDA level did not significantly

alter in other brain regions.

increased the MDA level in the corpus striatum (153% significantly

of control)

of

The combination

of AE + Et significantly

control) (F = 34.5, pLO.02) while it did not

change in other brain regions (Table 4).

Discussion This is the first report delineating

the combined effect of acute exercise (physical stress) and ethanol

ingestion (chemical stress) on ChE activity in blood and brain regions of the rat and their relationship lipid peroxidation.

The data indicate that acute exercise alone significantly

to

increased BuChE activity in

plasma and decreased AChE activity in the corpus striatum without affecting other brain regions.

BuChE

Exercise,

is a nonspecific cholinesterase

ethanol/cholinesterase

which is synthesized

665

in the liver (Augustinsson

and Nachmansohn,

1949).

Physical exercise is a stress factor that is known to evoke a number of metabolic and enzymatic changes in the liver and muscle (Booth and Thompson.

1991; Davies et al, 1982). Thus, the increased plasma

BuChE activity may be a secondary effect of labilization of lysosomal membranes of liver cells and leakage of enzyme in the blood after exhaustive exercise.

The present findings are consistent with other reports

(Pawlowska et al. 1985; Ryhanen et al. 1988).

Table 3 Effect of Acute Exercise (AE), Ethanol (Et) and AE + Et on Lipid Peroxidation Brain Regions of the Rat.

(MDA) in Plasma and

Lipid Peroxidation (Malondialdehyde)’ Brain Regions Cerebral Cortex

corpus Striatum

Medulla

Cerebellum

Hypothalamus

Plasma

157.03*14.47

240.83*41.15

237.25128.24

130.4317.7

67.74+6.11

53.61&6.75

2. Acute Exercise (n=4)

206.61c*l 1.68 (+32%)

510.57p&38.68 (+I 54%)

329.55’+38.68 (+39%)

140.37*17.72 (+8%)

78.67i4.92 (+16%)

60.54h2.94 (+7%)

3. Ethanol (n=4)

202.04*50.68 (+29%)

316.80c’di20.10 (+32%)

304.40*3 1.64 (+28%)

137.25h8.70 (+5%)

77.12il3.71 (+14%)

42.6li6.06 (-2 I o/b)

4. Acute Exercise + Ethanol (n=5)

185.851t26.09 (+lS%)

367.74b’e*10.13 (+53%)

273.82+6.88 (+15%)

148.48*5.39 (+14%)

79.39i12.94 (C17%)

56.20+8.75 (+5%/o)

Groups I. Sedentary Control (n=6)

Data are mean f SEM; * - nmoles MDA/g tissue or ml of plasma; ’ - significant (p < 0.01) compared with groupl;bsignificant (p < 0.02) compared with group 1; ’ - significant (p < 0.05) compared with group 1; d - significant (p < 0.01) compared with group 2; e - significant (p < 0.02) compared with group 2; Values in parentheses indicate percent change (+) increase and (-) decrease

K. Husatn and SM. Somani

666

Table 4 Correlation of AChE Activity with Lipid Peroxidation

in Different Brain Regions of Rat

AChE Activity Brain Regions

AE

Et

Medulla

I

1

Cerebellum

I

I

Lipid Peroxidation AE+Et

(MDA)

AE

Et

tt

t

f

I

T

r

I

1

T

AE+Et

Cortex Striatum

I

t Hypothalamus 1 Tor 1 = increase or decrease is not significant;

T

1T or 1I = increase or decrease is significant

AE = Acute Exercise’ Et = Ethanol

Conversely, AChE activity decreased only in the corpus striatum, the region responsible motor functions, after acute exercise. regions of the brain which

Interestingly,

for controlling

the data show the pattern of AChE inhibition in sub-

is well correlated with a significant increase in lipid peroxidation.

possible that this could lead to changes in synaptic membrane fluidity and permeability.

It is quite

The differences

observed in the regional distribution of AChE are consistent with the known cholinergic innervation to the brain regions that have been previously examined (Appleyard et al. 1986; Eckstein et al, 1988; Husain and Vijayaraghavan.

1989; Matin and Husain, 1985). Under normal physiological

conditions,

tissue levels of

acetylcholine (ACh) are regulated by the net synthesis and degradation of ACh by choline acetyltransferase and AChE. respectively.

Following exhaustive exercise, striatal levels of ACh rise with AChE inhibition

due to increased lipid peroxidation. exercise showed regional selectivity, is involved acetylcholine

in controlling

specifically

motor activity

elicited by acute

for the corpus striatum (deeper area of the brain) which

(Benarroch

et al. 1986; Matin and Husain,1985),

rate (Matin and Husain. 1985; Somani et al., 1996). However, AChE and lipid

in various brain regions have been reported to be altered differently

conditions (Appleyard et al. 1990; Estevez et al, 1984; Tsakiris and Kondopaulos. The data indicate that ethanol significantly

to different

activity

in the cerebellum

stress

1993).

decreased AChE activity only in the corpus striatum while

it slightly increased enzyme activity in the cerebral cortex. medulla and hypothalamus enzyme

where

has potent actions (Matin and Husain. 1985). This region is also rich in AChE and has a

higher lipid peroxidation peroxidation

The changes in AChE activity and lipid peroxidation

indicating

differential

response

within sub-regions

without altering of the brain.

Interestingly. changes in AChE activity in specific brain regions correspond to increased lipid peroxidation. A single dose of ethanol is known to increase lipid peroxidation

in the brain (Bondy, 1992; Burmistrov et

al, 1992; Bykova and Zhukova, 199 1; Uysal et al, 1989), and affect the cholinergic

system (Arendt et al,

Exercise, ethanol/chohnesterase 1988; Hashemzadeh-Gargari

667

and Mandel, 1989; Soliman and Gabriel, 1985).

ACNE activity may be suggestive of an altered acetylcholine

The inhibition of striatal

level and thereby altered motor control with

ethanol intake. These behavioral and biochemical deficits have been known to be caused by ethanol intake (Lishman,

1990; Rawat,

1976).

determining the above deficits.

However,

and Flevora,

investigators.

1982).

and inhibit AChE activity in rat brain (Burmistrov

Thus, the present

data are consistent

In vitro studies have also revealed perturbation

membrane-bound

in

The low acute and chronic doses of ethanol (1 .O and 1.5 g/kg) have been

shown to increase lipid peroxidation Sytinskii

the dose and duration of ethanol intake is important

AChE activity

with the findings

of the synaptic membrane,

at 40-60 mM ethanol concentration

et al, 1992; of the other

and changes in

(Lasner et al, 1995; Sun and

Samorajski, 1970) with no significant change in soluble form of AChE activity indicating the involvement of the membrane membrane-bound a protein-lipid

in ethanol action.

Ethanol (200-600 mM) caused a greater level of inhibition

in

AChE enzyme than in soluble enzyme in vitro (Baker and Chen, 1989) indicating that

interaction is needed to maintain the conformation

inhibition of membrane-bound

of membrane-bound

AChE.

Thus, the

AChE with increased membrane lipid peroxidation due to ethanol ingestion

in rats. as observed in the present study, is consistent with the in vitro study. Ethanol consumption humans (Blomquist

has been reported to adversely influence the treadmill exercise performance

in

et al, 1970; Houmard et al, 1987; Kendrick et al, 1993; Side11 and Pless, 1971).

However. other studies did not support these findings (Bobo, 1972; Mazess et al, 1968), which may be due to differences in the intensity. duration, type of exercise and the dose of ethanol used in these studies. data show that the combination

of exercise and ethanol 20% (1.6 gmikg, p.o.) also selectively

striatal AChE activity and increased lipid peroxidation

indicating influence of the combination

Our

decreased on motor

activity. The sensitivity of this brain region to the combination of other stressors has also been reported in the rat (Pal et al, 1993). Thus, ethanol intake after acute exercise partially modified the striatal AChE activity and lipid peroxidation exercise-induced

which may also partially improve motor function.

Ethanol decreased

increases in plasma BuChE activity suggesting that ethanol reduces the stress response

of exhaustive exercise at peripheral site.

Conclusions Acute exercise, single ethanol (1.6 gm/kg, p.o.) intake and the combination selectively inhibited striatal AChE

activity

perturbation

and the inhibition

of motor activity.

was correlated The combination

with an increase decreased

in lipid peroxidation

plasma BuChE indicating

suppresses the peripheral stress response caused by exhaustive exercise.

indicating that ethanol

666

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Inquiries and reprint requests should be addressed to:

Satu M. Somani, Ph.D. Professor of Pharmacology and Toxicology Southern Illinois University School of Medicine Department of Pharmacology P.O. Box 19230 Springfield, IL 62794- 1222 USA