Toxicology Letters, 51 (1990) 109-l 16

109

Elsevier

TOXLET

02305

Inhibition of rat brain microsomal Na+/K+-ATPase and ouabain binding by mercuric chloride

Chellu S. Chettyl,

Bettaiya Rajanna’

and Sharada

Rajanna2

1Department qf Natural Sciences and “Department of Computerand Mathematics. Selma University, Selma, AL (U.S.A.) (Received

30 June 1989)

(Accepted

29 October

Key words: Mercuric

1989) chloride;

Ouabain;

Na+/K+-ATPase;

Microsomes

SUMMARY This study concerned rat brain microsomes cromolar

concentrations.

and incubation

Cumulative

chloride

The degree of inhibition

time. Variations

of Na+/K+-ATPase of [)H]ouabain

the effects of mercuric

on Na+/K+-ATPase

in vitro. The data showed that HgC12 inhibited

activity by HgCIZ. Repeated

to microsomal

inhibition

was decreased

in the ionic strength membranes

washings

was inhibited

studies with HgClz and ouabain

ently and independently

of Na’

and [‘Hlouabain

Na+/K+-ATPase

with increases

binding

effectively

in enzyme concentration

and K+ did not alter the percent

partially

inhibition

enzyme activity.

The binding

by HgC& in a concentration-dependent

manner.

indicated

restored

in

at mi-

that these inhibitors

did not act concurr-

on Na+/K+-ATPase.

INTRODUCTION

Mercuric compounds are known to alter membrane-bound ATPase in a variety of tissues both in vitro and in vivo [1,2]. Na+/K+-ATPase is the molecular and enzymatic form of the (Na,K) pump which actively transports Na+ intracellularly and Kf extracellularly across the cell membrane [24]. This enzyme is central to normal brain function and its impairment would lead to widespread metabolic derangement. Ouabain. a cardiac glycoside, is known to inhibit Na+/K+-ATPase specifically by binding at catalytic sites [5]. Ouabain’s inhibitory effect on Na+/K+-ATPase is Na+dependent and irreversible [6,7] whereas the effect of mercuric chloride was both Na+- and K+-dependent and reversible [8]. The present investigation was therefore Address of correspondence: Prof. B. Rajanna,

Department

of Natural

Sciences,

Selma University,

AL 36701. U.S.A.

0378-4274/90/$3.50

@ 1990 Elsevier Science Publishers

B.V. (Biomedical

Division)

Selma,

initiated to study the in vitro effects of HgCIz on Na’/K’-ATPase and [3H]ouabain binding in rat brain microsomes. Also, independent and additive actions of HgCll and ouabain on Na+/K+-ATPase were studied to see whether there is any interference between

these two inhibitors

MATERIALS

of Na+/K+-ATPase.

AND METHODS

Male Sprague-Dawley rats (I 75-200 g) were obtained from Harlan Sprague Dawfey Inc., Indianapolis. IN. All biochemical agents used for enzyme assays were obtained from the Sigma Chemical Co.. St. Louis, MO. Radioactive labelled ouabain was obtained from New England Nuclear Corporation, Boston, MA. The rats were killed by decapitations the whole brain was homogenized in 9 vol. of ice-cold 0.32 M sucrose solution (pH 7.5) containing 10 mM imidazoie and I mM EDTA. Microsomes were prepared as described by Koch [9]. The homogenate was centrifuged for 10 min at 900 x g followed by centrifugation for 20 min at 12 000 x g. The microsomal membrane particles were sedimented from the 12 000 x g supernatant by centrifugation for 60 min at 100 000 x g. The microsomal pellets were resuspended and appropriately diluted in the ice-cold sucrose solution. quick-frozen in liquid nitrogen, and stored at - 85’:‘C. Microsomal Na+ jK “-ATPase activity was measured using end-point phosphate analyses [lo]. A l-ml reaction mixture was used and contained in final concentration 5 mM ATP, 5 mM MgC&, 100 mM NaCl. 20 mM KCI. 135 mM imidazole-HCI buffer (pH 7.5), and 30-35 pg of enzyme protein. Total cation ligand-stimulated ATPase activity was measured with Na+, K+ and Mg ‘+ in the reaction mixture. Mg’+ATPase was measured in the presence of 1 mM ouabain, a specific inhibitor of Na’j K+-ATPase. Thus, delineation of the (Nai.~K+)-activated component of ATPase was obtained by determining the difference between total ATPase and MgZi -ATPase. Incubation was carried out at 37°C for 30 min; the reaction was stopped with trichloroacetic acid at a final concentration of 5%. Samples were assayed for inorganic phosphate (Pi), using the method of Lowry and Lopez [l l] as modified by Phillips and Hayes [12]. Enzyme activity was expressed as Jlrnol Pi formed per mg protein per h. The effect of HgCl2 was assessed by preincubating the microsomes for about 5 min before, the reaction was initiated by ATP. Protein was determined by the method of Lowry et al. [ 131,using bovine serum al bumin as the standard. To study the reversibility of inhibition of Na+/K+-ATPase, 3 enzyme preparations were diluted in sucrose solution and after removing aliquots (containing 30--35 pug protein/50 ~1) for control assay, the remaining protein was treated with 2.0 x lo-’ M of HgClz and aliquots were removed for treated assay. Control and treated protein samples were diluted IO-fold with sucrose solution and centrifuged at 100 000 x g for 1 h. The washing procedure was repeated twice. Control pellets were assayed for activity before and after the complete washing procedure. The binding of [3H]ouabain to rat brain microsomes was determined by using the

111

method

of Schwartz

ml contained

et al. [14] with slight modifications.

The reaction

mixture

5 mM ATP, 5 mM MgC12, 100 mM NaCI, 50 mM Tris-HCl

of 2.0

(pH 7.4)

and 3540 pg microsomal protein. The reaction mixture was incubated for 15 min at 37°C after the addition of 0.1 ,uCi [3H]ouabain (1 mCi/0.049 mg). After incubation, the reaction

mixture

was filtered through

a 0.45~pm Millipore

filter and washed with

buffer. The filters were transferred directly to vials containing activity was determined using a liquid scintillation counter. a I-mM concentration was used for determining non-specific tracted from the total binding to obtain the precise amount to microsomes. Different concentrations of HgC12 were added as described previously.

10 ml Aquasol. RadioUnlabelled ouabain at binding. This was subof [3H]ouabain bound to the reaction mixture

Statistical analysis Each preparation was assayed in triplicate, and a mean of 3 or 4 different preparations was used to derive the standard error (SE) of the mean. The statistical significance of the difference between control and experimental values was calculated by Student’s f-test. A P-value of ~0.05 was considered significant. Cumulative effects of HgClz and ouabain in combination on Na+/K+-ATPase were calculated by the method of Woolfolk and Stadtman [15]. RESULTS

Na+/K+-ATPase in rat brain microsomes was inhibited by HgC12 in a concentration-dependent manner with an estimated IC 50, of 2.37 x 10V7 M HgC12 (Table I). At 15 pg microsomal protein, linear rates of ATP hydrolysis without significant differences were observed in inhibition by HgCl2 throughout 45 min incubation time. However, at 50 pg of microsomal protein, no such linearity was observed, and the percent inhibition TABLE

of enzyme activity by HgC12 was significantly

I

EFFECTS

OF HgClz ON MICROSOMAL

Na+/K+-ATPase

IN RAT BRAIN

HgClz

Specific activity

WI)

(,umol P,/mg protein/h)

0.0

13.5io.19

0.1

11.0~0.61

0.2

8.6+0.19*

0.3

4.0 * 0.07*

0.4

1.6_+0.13*

0.5

1.4+0.09*

*Significantly

different

from control

(PcO.05)

Each value is mean k SE of 4 different

preparations,

each assayed

in triplicate.

decreased

(Table II).

TABLE

II

EFFECT

OF ENZYME

OF RAT BRAIN ^____~_. Enzyme

CONCENTRATION

MICROSOMAL

TIME

ON HgC& INHIBITION

time (min)

15

(Pug)

INCUBATION

_...

Incubation .-..

COllC.

AND

Na+;‘K+-ATPase

30

--__

45

Cont

Expt

Cont

Expt

Cont

3.1

0.4 ,0.07*

5.7 &IO.29

I .o

8.3

_+0.08”

kO.51

_.-___I____._..

I5

*0.07

(87.1) 50

Expt _. .._.. 1.2

(X2.5)

12.1

6.9

15.1

11.2

15.1

+0.55

+0.X*

kO.07

*0.07*

kO.62

(42.8,

& 0.03* (85.51

(15.5)

14.9 kO.51 (1.26)

_._” __~~ *Significantly

different

from control

Each value is mean *SE rate percent

decreases

(PC 0.05).

of 4 different

over control.

preparations

Cont =control:

each assayed

in triplicate.

Values in parentheses

indi-

Expt =experimental.

At optimal Nai concentration (100 mM), K* varied from 0.5 to 20 mM, while at optimal K’ (20 mM) Nat varied from 1 to 50 mM. No significant differences were observed in percent inhibitions of Nat/K’-ATPase activity at both lower K ’ and N’a+ concentrations (Table III). Reversibility of Na+/K+-ATPase inhibition by TABLE

III

INHIBITION

OF Na+/K+-ATPase

ED ACTIVITY

BY HgC& (2 x 10~’ M) AT VARYING

NaCl

KC1

Na’/K

(mM)

(mM)

(s inhibition) ~_. __. __~~_.

100

u.so

71.1 k3.81

100

1.50

69.6+4.2* 69.4+ 3.5*

I 00

2.50

100

3.50

71.02

100

5.00

69.

100

‘-ATPase

1.4*

10.00

70.7-i: 1.4*

20.0

81.3+1.1*

2.5

20.0

74.91

5.0

76.8*0.3*

10.0

20.0 20.0

69.6+2.8*

25.0

20.0

74.8& 1.2*

50.0

20.0

76.011.1*

“Values are means

&SE percent inhibitions

significant

(PCO.05).

of 4 independent

activity*

I * I .4*

1.0

*Statistically

CATIONIC-STIMULAT-

STATES

i.t*

samples each assayed

in triplicate.

113 TABLE

IV

REVERSIBILITY

OF INHIBITION

Experimental

Specific

treatment

activity

OF Na+/K+-ATPase

BY HgC12 WITH WASHING

% Inhibition

% Recovery”

cum01 P,/ mg protein/h) Control

16.18~1.10

Treated

4.63 _+0.22*

71.4

Wash I

6.46 +0.57*

60.1

11.3

Wash 11

7.49+0.33*

53.7

17.7

Wash III

7.82 +0.08*

51.7

19.7

“Obtained

after subtracting

*Significantly

different

from treated

from control

sample value.

(P< 0.05).

Each value is mean k SE of 3 different

enzyme preparations

each assayed

in triplicate.

HgClz was demonstrated by repeated washings of the enzyme preparation. After 3 separate washings, centrifugations, and resuspensions of treated enzyme aliquots, 19.7% of original activity was restored (Table IV). The binding of [3H]ouabain to microsomes in vitro was determined in the presence of different concentrations of HgC12 (Table V). The binding decreased in a concentration-dependent manner when up to 0.75 ,uM HgC12 was preincubated with the microsomes. A maximum decrease was observed with increase in HgClz concentration to 1.0 PM. The estimated ICsO for HgClz was found to be 3.5 x lo-’ M. The different concentrations of ouabain in combination with 2.0 x lo-’ M HgC12 were used to assess their independent and additive effects on Na+/K+-ATPase. The data (Table VI) show that both HgC12 and ouabain inhibited the Na+/K+-ATPase activity indivi-

TABLE EFFECT

V OF HgC12 ON [ZH]OUABAIN

BINDING

TO RAT BRAIN

HgC12

[3H]Ouabain

(PM)

(cpm/mg

517& 17.5

0.05

4545

0.25

239* 12.1*

0.50

153* 10.2*

12.2*

0.75

57*7.0*

I .oo

30+2.9* different

Each value is mean

from control

binding

proteimmin)

0.0

*Significantly

MICROSOMAL

(PC 0.05)

+SE of 4 individual

observations

each assayed

in triplicate.

FRACTIONS

I I3

TABLE

VI

CUMULATIVE

EFFECTS

OF OUABAIN

Inhibitor

AND HgClz ON Na’;K+-ATPase Na+jK

‘-ATP-

Observed

Calculated

ase

inhibition

cumulative

activity

(5)

inhibition (5)

Control

(no inhibitors)

26.3 iO.2

HgCl?

2.0x10

‘M

13.7*0.2*

47.9

Ouabain

1.0~10

hM

13.1*0.3*

50.2

Ouabain

1.0x IO-‘M

16.8*0.8*

36.1

Ouabain

1.0~ 10mXM

l8.5k

39.6

Ouabain

1.0x IO-“M

+ HgCI? Ouabain

2.0x

+ HgCl2 Ouabain

2.0 x IO-’ M

+ HgCl:

3.0x

*Statistically

10

1.0~10 1.0x10

significant

Each value is mean *SE

iM

1.7*

10.9&0.5*

58.5

78.3

12.6f0.5*

53.1

75.0

13.3+0.7*

49.4

73.6

‘M XM

IO-‘M

(P

K(+)-ATPase and ouabain binding by mercuric chloride.

This study concerned the effects of mercuric chloride on Na+/K(+)-ATPase and [3H]ouabain binding in rat brain microsomes in vitro. The data showed tha...
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