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