A CELLULAR ACTION OF MERCURIAL DIURETICS’

A CELLULAR ACTION OF MERCURIAL DIURETICS’

23

VERNON D. JONES, GEORGE LOCKETT AND ERWIN J. LANDON

Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee

Accepted for publication October 2, 1964

Mercurial diuretics block the transport of

sodium ions across renal tubular cells. This

action has been attributed either to direct in-

hibition of carriers for sodium ions or to in-

hibition of the energy supplied to the sodium

transport system (Pitts, 1962). The ability of

these compounds to inhibit sodium reabsorption

by the renal nephron has not been correlated

satisfactorily with their effects on biochemical or

biophysical events involved in the reabsorptive

process. It is the purpose of the present paper

to demonstrate a cellular action of the mercurial

diuretics that correlates in vivo and in vitrowith their diuretic action.

A considerable body of experimental evidence

suggests that the (Na� and K�)-dependent

ATPase’ activity found in red blood cell and

kidney membranes is a property of the coupled

transport system for sodium and potassium in

the membrane (Post et a!., 1960; Landon and

Norris, 1963). This ATPase system in kidney

membranes is inhibited in vitro by diuretic

mercurials (Landon and Norris, 1963; Taylor,

1963). Histochemical studies indicate that the

kidney ATPase activity is localized primarily

in the membranes of the peritubular region

(Spater et at., 1958; Ashworth et at., 1963)

where active sodium transport takes place

(Giebisch, 1962).

The ATPase activity of a membrane frac-

tion, isolated from rat kidney, has been shown

to stimulate the phosphoglycerate kinase reac-

tion of cytoplasmic glycolysis (Jones et at.,

1963). The membrane ATPase hydrolyzes the

ATP formed by this step of glycolysis, driving

the glycolytic pathway in the forward direction

Received for publication July 31, 1964.

1This research was supported by grants from

the United States Public Health Service (AM08649 and AM 04703) and the American CancerSociety (IN-25C).

‘ADP, adenosine diphosphate; ATP, adenosinetriphosphate; ATPase, adenosine triphosphatase;ER, endoplasmic reticulum; FDP, fructose-i ,6-diphosphate; NAD�, nicotinamide adenine di-nucleotide; PCMB, p-chloromercuribenzoate;PCMS, p-chloromercuriphenylsulfonate and 5,,cell sap (cytoplasm).

and increasing the rate of both the kinase re-

action and overall glycolysis. The membrane

stimulation of glycolysis was not simply a con-

sequence of enhanced ADP levels maintained

by the membrane ATPase activity. The same

stimulation occurred at ADP levels four times

greater than that required for optimal glycoly-

sis. Because cellular membranes are contiguous

with cytoplasm containing glycolytic enzymes,

this interaction of membrane and glycolysis is

interpreted to be a physiological link between

energy metabolism of the cell and active sodium

and potassium transport in the membrane

(Jones et at., 1963).

The present study reports the effect of mer-

curial diuretics, in vitro and in vivo, on the

ATPase system of this membrane fraction and

the interaction of the ATPase system with cyto-

plasmic glycolysis. When subcellular fractions

are prepared from rats pretreated in vivo with

organic mercurial compounds, only the diuretic

mercurials inhibit the ATPase system of this

membrane fraction. The data are in agreement

with the concept that mercurial diuretics in-

hibit active sodium reabsorption in vivo by in-

hibiting the (Na� and K�)-activated component

of the membrane ATPase system. A prelimi-

nary report of these findings has been published

(Jones and Landon, 1963).

METHODS. Preparation of tisrue fractions. Kid-

neys from male Sprague-Dawley albino rats, 3 to

4 months old, were used in this study. The ratswere allowed free access to food (Purina labora-

tory chow) and water at all times. The kidneyswere removed immediately after sacrificing the

animals, placed in 0.25 M sucrose at 0#{176}C,and keptat this temperature throughout the preparativeprocedure (Landon and Norris, 1963). An endo-

plasmic reticulum (ER) fraction and cell sap (83)

fraction were obtained by differential centrifuga-tion of a kidney homogenate (2 ml 025 M su-crose/g kidney). The ER fraction is that part ofthe homogenate which sediments during centrifu-

gation between 37,000 X g for 40 minutes and105,000 X g for 120 minutes. The sediment was

then resuspended in the original volume of 0.25 M

24 JONES ET AL. Vol. 147

sucrose. The supernatant of the 120-minute spin

is the cell sap fraction. Both fractions were quick-frozen in a dry ice-ethanol bath (-60#{176}C) and

stored at -20#{176}C.The isotonic sucrose employed inthis preparative procedure contained either 1 or 2

mM EDTA.Chemicals and enzyme assays. Fructose-i , 6-

diphosphate (FDP), p-chloromercuribenzoate(PCMB),p-chloromercuriphenylsulfonate (PCMS),

adenosine 5’-diphosphate (ADP), adenosine 5’-tri-phosphate (ATP) and nicotinamide adenine di-

nucleotide (NAI)1 were obtained from SigmaChemical Co. Meralluride was kindly provided byLakeside Laboratories. Thiomerin (mercaptomerin)

and Mercuhydrin (meralluride and theophylline)

were obtained from the hospital pharmacy.

ATPase (adenosine 5’-triphosphatase) activity

of the ER fraction was assayed by incubating 02

ml of the enzyme preparation in 4 mM Mg-ATP,

33 mM Tris buffer (pH 72) with and without 133

mM NaC1 and 33 mM KC1, at 37#{176}Cin a Dubnoff

metabolic incubator shaking at 80 cycles/minute.The final volume was 3.0 ml. After a 20-minute

incubation, 1-ml aliquots were deproteinized with

1 ml of 5% cold trichloroacetic acid. Inorganic

phosphate liberated by the ATPase reaction was

measured by the method of Lowry and Lopez

(1946). Protein of the ER fraction was deter-

mined by a modification of the phenol method

using crystalline bovine albumin as the protein

standard (Sutherland et at., 1949). Results of the

assay are expressed as micromoles of inorganic

phosphate liberated per mg of enzyme protein.

Glycolytic activity of the cell sap (Se) fraction

was determined under aerobic conditions (open

air) at 37#{176}Cin a Dubnoff incubator shaking at 80cycles/minute. The assay medium contained 50

mM potassium phosphate buffer (pH 7.0), 3 mM

MgCI2, 1 mM ADP, 1 mM ATP, 20 mM nico-

tinamide, 0.05 mM NAD�, 10 mM FDP and 0.3

ml of the S3 fraction. Three-tenths ml of the ER

fraction was added where the influence of the ER

fraction on glycolysis was being measured. Con-

trol incubations in these experiments received in-

stead 0.3 ml of 025 M sucrose containing 1 mM

disodium EDTA. The final volume of the incuba-

tion mixture was 3.0 ml. Each incubation was for

60 minutes. All solutions were adjusted to pH 7.0

before use. Lactate formed during glycolysis was

determined by the procedure of Barker and Sum-

merson (1941) and pyruvate formed during gly-

colysis by the procedure of Friedman and Haugen

(1943).

Chlorides were determined by the method of

Schales and Schales as cited by Smith (1956).

RESULTS. The effect of organic mercuriaLs

on a membrane ATPase system. The ATPase ac-

tivity of a freshly prepared endoplasmic retic-

ulum fraction (ER) was not stimulated by the

addition of Na� and K� (Landon and Norris,

1963). However, if the fraction was dialyzed (to

remove the bound Na� and K�), the ATPase

activity was then reduced about 50%. This ac-

tivity could be restored to a large extent by

adding Na� and K�.

Meralluride had little or no effect on the

ATPase activity of dialyzed preparations in the

absence of added Na� and K�. The (Na� and

K�)-activated ATPase system of the dialyzed

ER fraction was inhibited by low levels of

meralluride. Higher levels of meralluride were

required to inhibit ATPase activity in non-

dialyzed preparations (fig. 1). The meralluride-

inhibited ATPase activity of nondialyzed prepa-

rations appears to be the ATPase activity which

is associated with bound sodium and potassium.

It has been postulated that the Na� and K�

which are bound to the nondialyzed preparations

partially protect the membrane (Na� and Ki-

activated ATPase system from inhibition by the

mercurial diuretics (Landon and Norris, 1963).

Results similar to those obtained with meral-

luride were usually obtained with equivalent

concentrations of mercaptomerin.

The nondiuretic mercurials PCMB (p-chloro-

mercuribenzoate) and PCMS (p-chloromercuri-

phenylsulfonate) (Miller and Farah, 1962;

Weiner et a!., 1962) also exhibited inhibitory

effects on the ATPase activity of the ER. At

lower levels, however, these two compounds ap-

peared to stimulate the ATPase activity. These

opposing effects were clearly separated in ex-

periments with dialyzed ER fractions. Figure

2 represents a typical experiment in which vari-

ous levels of PCMS were added to the ATPase

assay medium containing dialyzed ER, both in

the presence and absence of Na� and K�. At low

levels of PCMS the magnesium-dependent ATP-

ase activity was increased, while the (Na� and

K�) -activated component of the ATPase system

was inhibited at all levels of PCMS (as shown

by the bottom curve). A stimulation of the

ATPase similar to that seen with low levels of

PCMS was occasionally seen with low levels of

mercaptomerin.

The effect of organic mercurials on the inter-

action of the ER and cell sap fraction. It has

-4’-- Dialyzed ER+33mM NaCI

+ 33mM XCI

..._�Dialyzed ER

.-o�Difference

C

4’00.

Ui

EC

E

I

9

8

7

6

5

4

3

2

0.� 9.

4)

�I.

I I I I I � .�-I

00.1 0.3 0.6 I 2 10

[Meralluride] X I04M

FIG. 1. The effect of various levels of merallurideon the ATPase activity of the endoplasmic reticu-lum (ER) membrane fragments.

Assay conditions are described in the section onMETHODS. Preparations that were dialyzed for 18hours at 2 to 4#{176}Cwere assayed in the presence of133 mM NaCl and 33 mM KC1. The nondialyzedER curve represents the mean of 10 experiments.The dialyzed ER curve represents the mean of 5experiments. The vertical bar represents the stand-ard errors of the means. The dashed line repre-sents the (Na� and K�-independent ATPase ac-tivity estimated as approximately 50% of the totalactivity.

1965 MERCURIAL DIURETICS AND ATPASE 25

been reported previously that glycolytic activity

of the cell sap (S3) fraction is enhanced by ad-

dition of the ER fraction (Jones et al., 1963).

This stimulation of glycolysis is due to the ATP-

ase activity found in the ER membrane. Since

organic mercurials inhibit the ATPase system,

the effect of these agents on the stimulation of

glycolysis by the membrane system was studied.

Table 1 shows that, as increasing amounts of

meralluride were added to the assay medium,

the stimulation of glycolysis by the ER frac-

tion was progressively diminished. The stimula-

tion of glycolysis by the ER fraction was 32%

in the presence of 10’ M meralluride and 91%

stimulation when no mercurial was added. In

the absence of the ER fraction the glycolytic

activity of the cell sap fraction was not sig-

nificantly altered by the presence of 10� M

meralluride. Mercaptomerin (table 1) caused

a reduction of the stimulation of glycolysis by

the ER fraction in a manner similar to meral-

luride. The nondiuretic mercurials PCMB and

PCMS in vitro also reduced the stimulation of

glycolysis by the ER fraction (table 1).

31

Non-dialyzed ER��yzedER + I33mMNaCI

+ 33mMKCI

--- -

I I I I I�F

00.1 0.3 0.6 1.0 2.0

(PCMS) X 104M

Fio. 2. The effects of various levels of p-chloro-mercuriphenylsulfonate (PCMS) on the ATPaseactivity of ER dialyzed for 18 hours at 2 to 4#{176}Cassayed in the presence and absence of 133 mMNaC1 and 33 mM KC1.

Assay conditions are described in the section onMETHODS. The bottom curve represents the Mg-ATPase activity that results from the presence ofNa� and K�.

The effect of mercurials on the stimulation

of glycolytic activity by the ER fraction paral-

leled the effects seen on the (Na� and K�)-acti-

vated component of the ATPase system of the

ER fraction. The per cent reduction of the ER

stimulation of cytoplasmic glycolysis was 65%

with 10� M meralluride and 67% with 10� M

mercaptomerin (table 1). Meralluride (10� M)

inhibited the (Na� and K�)-activated compo-

nent of the ATPase system of the dialyzed ER

fraction by 81% and the (Na� and K�)-activated

component of the nondialyzed ER fraction ap-

proximately 67% (fig. 1). The data suggest that

the stimulation of glycolysis is specifically

caused by that component of the membrane

ATPase system which is activated by Na� and

K�.

The effect of mercuric chloride on the ER-

cell sap interaction. The effect of adding vari-

ous levels of mercuric chloride on the coupled

ER-cell sap glycolyzing system is shown in

figure 3. Mercuric chloride inhibited the glyco-

lytic activity of the cell sap and the stimulation

by the ER fraction proportionately. We have

also confirmed the results of Taylor (1963) who

observed that mercuric chloride inhibited non-

specifically both the (Nat and K�)-activated

The in vitro effect

Meralluride

TABLE 1

of organic mercurials on glycolytic activity” of the rat kidney cell sap fraction

Mercaptomerm p-Chloromercuri-benzoate

MercurialConcentration

0

0.3 X 10-4 M

0.6 X 1O-�M

1.0 X 1O�M

2.0 X 1O4 M

p-Chloromercuri-phenylsulfonate

Tissue

51b

Ss+ ER

SI

S*+ ER

SIS�+ ER

SI

Ss+ ER

SI

Ss+ ER

Lactate(1iniol/hr)

17.4 ± 1.3 (9)C

33.3 ± 2.6 (9)

17.8 ± 2.0 (6)

27.6 ± 3.1 (6)

17.7 ± 1.1 (9)

25.2 ± 1.9 (9)

15.8 ± 1.6 (6)d

20.8 ± 2.2 (6)

%Increaseby ER

fraction

91

55

42

32

Lactate(j�mol/hr)

14.5 ± 0.6 (8)31.9 ± 1.8 (8)

14.8 ± 0.8 (7)

28.4 ± 1.4 (7)

14.6 ± 0.4 (6)

24.6 ± 0.7 (6)

13.6 ± 0.4 (6)d

19.0 ± 1.2 (6)

11.2 ± 1.1 (6)

15.4 ± 1.5 (6)

%Increaseby ER

fraction

120

92

68

40

28

Lactate(j�mol/hr)

15.6 ± 1.3 (7)

31.8 ± 3.6 (7)

15.6 ± 1.0 (7)

26.6 ± 1.6 (7)

15.7 ± 1.1 (7)

23.5 ± 1.2 (7)

12.8 ± 1.0 (7)d

17.8 ± 1.1 (7)

8.1 ± 0.9 (7)

10.9 ± 1.1 (7)

%Increase

by ERfraction

104

70

50

39

22

Lactate(jimol/hr)

15.1 ± 1.3 (5)

27.9 ± 1.4 (5)

16.1 ± 0.4 (5)

22.9 ± 1.9 (5)

14.1 ± 0.5 (5)

17.4 ± 1.0 (5)

12.7 ± 0.8 (5)d

14.5 ± 0.8 (5)

%Increase

by ERfraction

85

42

23

14

#{176}Glycolytic assay conditions are described in the section on METHODS.b � denotes assay of the cell sap fraction and Si + ER denotes assay of the cell sap fraction to which ER membrane fraction has

been added.Mean product values are given ± the standard errors of the means. The figures in parentheses refer to the number of experi.

rnents.d Not significantly different from control (5% level).


and the (Na� and K�)-independent ATPase sys-

tem of the membrane. These data are consistent

with the existing concept that mercuric chloride

causes diuresis as a result of its nephrotoxic ef-

fects. Inhibition of glycolysis in the cytoplasm,

as well as inhibition of that component of

glycolysis which is activated by the cellular

membranes, would be expected to cause severe

toxicity to the cell.

The effect of pH on the ER-cell sap inter-

action. It is well established that the mercurial

diuretic effect is increased by prior administra-

tion of acidifying agents and decreased by the

administration of alkalizing agents (Ethridge

et a!., 1936; Levy et at., 1958). The experiments

in this study, carried out at pH 7, showed that

addition of the ER fraction stimulated glycolysis

and that meralluride inhibited this stimulation.

Experiments were performed in which the pH

of the medium was varied between 6 and 8 to

determine if change of pH affected the inhibi-

tion by meralluride. A level of meralluride was

chosen (0.6 x 10� M) that causes only partial

inhibition.

The glycolytic activity of the cell sap frac-

tion increased with an increase in pH. However,

with the pH range studied, the increase in

glycolysis caused by addition of the ER frac-

tion was fairly constant. Addition of meralluride

(0.6 x 10� M) resulted in a reduction of the

ER stimulation of glycolytic activity. The ac-

tivity lost, however, remained essentially un-

changed throughout the pH range employed.

This indicated that the meralluride inhibition of

the ER-cell sap interaction was not affected by

pH. These data did not explain the action of

acidifying agents on the diuretic action of or-

ganic mercurials.

Studies of tissues prepared from rats pre-

treated with organic mercurials. In the previous

experiments inhibitory effects were obtained

when organic mercurials were added to sub-

cellular fractions in vitro. Subsequent experi-

ments were designed to test whether in vivo

administration of organic mercurials to rats

would have a demonstrable effect on subcellu-

lar fractions prepared from their kidneys.

Mercaptomerin and Mercuhydrin were ad-

ministered intramuscularly to rats 2 hours prior

to sacrificing the animals for tissue prepara-

tions. The subcellular preparations from these

pretreated animals were assayed for ATPase

activity of the ER fraction and the ability of

the ER fraction to stimulate glycolysis of the

cell sap fraction. The same assay medium was

employed as in the control experiments. The

35�

FIG. 4. lkie effect of mercaptomerin pretreat-ment on the ability of the ER fraction to stimu-late cell sap (S3) glycolytic activity.

Assay conditions are described in the section onMETHODS. Vertical lines represent the standarderrors of the means. Mercaptomerin was adminis-tered intramuscularly 2 hours prior to sacrifice ofthe animals and preparation of the tissue frac-tions.

S3

�- S3+ER

.C

4’UU0

11)4’0

E

4’aU

a

4’00

E

1.0

FIG. 3. The effect of various levels of mercuricchloride on glycolytic activity of the cell sap frac-tion in the presence and absence of the ER frac-tion.

Assay conditions are described in the section onMETHODS. Each point represents the mean of 10experiments except at the 10� M level (6 experi-ments). Vertical lines represent the standard er-rors of the means.

[I1gcIg� xI0�M Mercuhydrin

Dose CmgHg

Number ofEaperiments

616 5

1% Increase

[by ER fraction 122 39 31

FIG. 5. The effect of Mercuhydrin pretreatmenton the ability of the ER fraction to stimulate cellsap (S8) glycolytic activity; the experimental de-sign was the same as that reported in figure 4.

1965 MERCURIAL DIURETICS AND ATPAsE 27

results from the coupled glycolytic experiments

are presented in figures 4 and 5.

As the dose of mercaptomerin or Mercu-

hydrin was increased, the stimulation of cell

sap glycolytic activity by the ER fraction di-

minished. The glycolytic activity of the cell sap

fraction alone was unaltered within the dose

range of 2 to 8 mg Hg/kg. At a higher dose

level of mercaptomerin (12 mg Hg/kg) the

glycolytic activity of the cell sap fraction was

inhibited slightly while the ability of the ER

fraction to stimulate glycolysis was further re-

duced.

The ATPase activity of nondialyzed ER frac-

tions prepared from pretreated animals was as-

sayed. The results are presented in figures 6

and 7. The ATPase activity diminished as the

dose level was increased. The per cent inhibi-

tion was very similar to that seen when the

mercurial diuretics were added in vitro.

Results from these experiments which em-

ployed the tissue preparations from animalspretreated with nondiuretic mercurials are pre-

sented in figure 8. Glycolytic activity of the cell

sap fraction was inhibited by PCMB at levels

of 4 or 8 mg Hg/kg. However, the stimulation

of cell sap glycolytic activity by the ER frac-

C4)900.

Ui

0i

E 7C

60(sJ�- 5.04)0 4.4)

.0

= 3.a-� 20E �.

I I

(9) (11) (5) (5) (5)

.C4’aU

aU,4’0E1*.

FIG. 8. The effect of p-chloromercuribenzoate(B) and p-chloromercuriphenylsulfonate (S) pre-treatment on glycolytic activity.

Shaded area represents glycolytic activity ofthe cell sap fraction. Open area represents addi-tional glycolytic activity in the presence of theER fraction. Experimental details are like thosein figure 4. Numbers on top of figure representnumber of experiments.

Mercurial Dose(mg Hg/kg)


tion was not significantly altered at these two

dose levels. Neither the glycolytic activity of

the cell sap fraction nor the ability of the ER

fraction to stimulate glycolysis was affected by

PCMS at levels of 4 or 8 mg Hg/kg. Thus,

the effects of nondiuretic mercurials in vivowere distinctly different from the effect of diu-

retic mercurials when tissue fractions were iso-

lated from pretreated rats. Neither compound

inhibited the stimulation of glycolytic activity

by the ER fraction.

The effect of pretreatment with nondiuretic

mercurials on the ATPase system of the iso-

lated ER fraction is seen in figure 9. Neither

PCMB or PCMS caused a decrease in ATPase

activity of the ER fraction at a level of 4 mg

Hg/kg. Both compounds were slightly inhibi-

tory at a level of 8 mg Hg/kg.

The diuretic response to mercurials. Experi-

ments were performed to determine the dose of

mercurial diuretic required to produce a sig-

nificant increase in both the urine volume and

* % Inhibition of estimated Na-K actIvatedMg-dependent ATPase activity.

FIG. 6. The effect of mercaptomerin pretreat-ment on the ER ATPase activity.

Assay conditions are described in the section onMETHODS. The experimental design was the sameas that reported in figure 1 employing nondialyzedER fractions. The open portions of the bars rep-resent the estimated (Na� and Ki-independentATPase activity, and the hatched portions repre-sent the (Na� and Ki-activated ATPase activity.The vertical lines represent the standard errors ofthe means.

Mercuhydrin - f T8 1Dose(mgHg/kg)

Numberof 251 6 6 1Experiments ___________________

%lnhibltion* - 69 �

* % Inhibition of the estimated Na-Kactivated ATPase activity

Fia. 7. The effect of Mercuhydrin pretreatmenton the ATPase activity of the ER fraction; seefigure 6 for experimental details.

(25) (10) (5) (5) (5)

0 4(8) 8(B) 4(S) 8(S)

Mercurial Dose (mgHg/kg)


the amount of chloride excreted. The results

are presented in table 2. Male albino Sprague-

Dawley rats received mercaptomerin or Mercu-

hydrin intramuscularly at dose levels of 4 and

8 mg Hg/kg. Six-hour urine samples were col-

lected and analyzed for chloride. The volume

and pH of the samples were determined also.

Significant diuresis was induced with mercapto-

merin or Mercuhydrin at 4 mg Hg/kg. The

diuretic effect was greatly increased by an 8-

mg Hg/kg dose. The dose required to get a sig-

nificant diuresis corresponded reasonably well

with the dose required to obtain significant in-

hibition of the ATPase system of the ER frac-

tion. Data are also presented in table 2 to show

that PCMB and PCMS at levels equivalent to

Mercuhydrin and mercaptomerin are not diu-

retic agents in the rat.

DISCUsSIoN. The present study shows that

the in vivo administration of diuretic doses of

organic mercurials inhibits activity of the (Na�

and K�)-dependent ATPase system in kidney

membranes and the ability of the membrane

ATPase system to stimulate glycolysis in the

cytoplasm. This cellular action of mercurial

diuretics could explain their pharmacological

action if the following criteria were met. The

altered ATPase system should be involved in

active sodium transport. The in vivo action of

C

a)00.

Uia.EC

E0c’,J04)0

a).0

a-U,a)0E

FIG. 9. The effect of p-chloromercuribenzoate(B) and p-chloromercuriphenylsuffonate (S) pre-treatment on the ER ATPase activity.

The experimental design is described in figure6. Numbers on top of figure represent number ofexperiments.

TABLE 2

The diuretic effect of mercaptomerin and

Mercuhydrin injected intramuscularly

into rats

6-Hr UrineVolume (ml)

otCon e H of

�Jrine

Control 4.8 ± 1.0 336 ± 31 6.8 ± 0.1Mercapto- 7.8 ± 0.6* 492 ± 58* 6.7 ± 0.2

merin (4 mg

Hg/kg)Mercuhydrin 9.5 ± 09* 584 ± 41* 6.6 ± 0.3

(4mg Hg/kg)Mercuhydrin 13.6 ± 1.6* 775 ± 62* 7.2 ± 0.2

(8 mg Hg/kg)

p-Chloro- 5.4 ± 0.6 317 ± 46 7.3 ± 0.2mercuriben-

zoate (8 mgHg/kg)

p-Chloro- 4.5 ± 0.9 250 ± 39 7.2 ± 0.3mercuri-phenylsul-

fonate (8 mg

Hg/kg)

* Significantly different from control at the 5%

level.Each value is a mean of 20 experiments ±

standard error except with PCMS (15 experi-ments).

organic mercurials on the ATPase system should

be limited to organic mercurials that are diu-

retics. The effect on the ATPase system should

be greater at acid pH than at a more alkaline

pH. The direct action of organic mercurials

described in this study appears to meet these

criteria.

The evidence for involvement of the kidney

membrane ATPase system in coupled transport

of sodium and potassium is considerable (Lan-

don and Norris, 1963; Landon and Forte, 1964).

The demonstrated inhibition of the membrane

ATPase system by mercurial diuretics both in

vitro and in vivo provides further evidence that

this ATPase system is involved in ion transport.

The inhibitory action of organic mercurials on

the membrane (Nat and K�)-dependent ATPase

is considerably less in nondialyzed membrane

preparations containing bound sodium and po-tassium than with dialyzed membranes from

which bound sodium and potassium have been

removed. This suggests that the bound ions pro-


tect the ATPase system from tile inhibitory ac-

tion of organic mercurials and that the organic

mercurials act by directly attacking sodium or

potassium binding sites of the transport sys-

tem.

In this study the inhibitory in vivo action is

limited to diuretic mercurials. Since in vitro

nondiuretic mercurials are also effective inhibi-

tors of the ATPase it must be assumed that in

vivo the nondiuretic mercurials are handled by

the kidney in such a way that they do not con-

centrate at the critical site of the ATPase sys-

tem.

The stimulation of glycolysis by the ER frac-

tion and the meralluride inhibition of this stim-

ulus were tested in this study between pH 6

and 8. Neither the absolute amount of stimula-

tion nor inhibition varied significantly with pH.

The (Nat and K�)-dependent ATPase activity,

however, increased considerably between pH 6

and 8 with increase in pH (Landon and Norris,

1963). Meralluride-inhibited preparations also

showed a similar increase in ATPase activity

with increase in pH (unpublished data). It

would appear probable that at lower pH levels

where the total activity of the ATPase system

is considerably less the mercurial inhibition

of the ATPase is more effective in bringing

about diuresis. Another possibility is suggested

by the observation of Cafruny that chlormero-

drin achieves a higher concentration in the

renal cortex of acidotic dogs (Cafruny, 1963).

It has been suggested (Weiner et al., 1962)

that intrarenal release of free mercuric ions

from organic mercurials by disruption of an

acid labile bond forms the basis of their diu-

retic action. The failure of the more acid pH to

enhance the absolute inhibition of the ATPase

system by organic mercurials does not seem to

be compatible with this concept.

The in vitro stimulation of glycolysis by the

ER membrane fraction is the result of the mem-

brane ATPase stimulating the phosphoglycerate

kinase reaction. Experiments with crystalline

phosphoglycerate kinase indicate that the mem-

brane ATPase utilizing ATP generated by phos-

phoglycerate kinase impedes the reaction pro-ceeding in the reverse direction (Jones et al.,

1963). It appears probable that in the intact

cell where cytoplasm and membranes are con-

tiguous a similar reaction takes place. The

experimental results in the present paper in-

dicate that only the (Nat aild K�)-dependent

component of the membrane ATPase activity

couples with phosphoglycerate kinase and stim-

ulates glycolysis. This evidence for a specific

coupling between the active transport ATPase

and phosphoglycerate kinase of the cytoplasm

lends further support to the concept that the

membrane-glycolysis interaction is a functional

adaptation facilitating the flow of metabolic

energy to the membrane.

In the medullary region of the kidney glycol-

ysis is considered to be the primary source of

energy for sodium transport (Kean et al., 1961).

K� accumulation in slices of rabbit kidney cor-

tex is supported by glucose or Krebs cycle

intermediates prior to succinate (Brendt and Le-

Sher, 1961). In the cortical region of the kid-

ney, the cytopiasmic phosphoglycerate kinase

reaction may be involved in the immediate sup-

ply of ATP to the membrane for active trans-

port. Mitochondria are abundant in the cyto-

plasm of the peritubular region of proximal

tubular cells. In the vicinity of the mitochondria,

uptake of ADP and release of ATP or phospho-

enolpyruvate into the cytoplasm would favor a

cyclic reversal of the phosphoglycerate kinase

reaction. The net amount of glycolysis or glu-

coneogenesis in the kidney would be independent

of this cyclic reaction. Diuretic levels of organic

mercurials which inhibit the membrane ATPase,

and as a consequence the membrane stimulated

glycolysis, do not inhibit mitochondrial respira-

tion in the kidney (Cohen, 1953; Fawaz and

Fawaz, 1954).

The in vivo and in vitro cellular action of

mercurial diuretics on the membrane ATPase

demonstrated in this study correlate well with

the diuretic action of the mercurials. The mer-

curials appear to act primarily on the ion bind-

ing sites of the ATPase transport system and as

a result of this action exert effects on glycolytic

energy metabolism. They appear to be more

effective at acid pH because of the overall de-

cline in ATPase activity at lower pH levels.

The data suggest that cytoplasmic glycolysis

may serve as the immediate energy source in

membrane transport.

SUMMARY

Diuretic and nondiuretic organic mercurials,

added in vitro, inhibit the (Na� and K�)-acti-

vated component of the ATPase system of mem-

brane fragments prepared from rat kidneys.


These compounds also reduce the ability of

the membrane preparation to stimulate cyto-

plasmic glycolysis. The (Na� and K�)-activated

ATPase activity was reduced when membrane

fragments were prepared from kidneys of rats

pretreated with therapeutic doses of diuretic

mercurials. The ability of these membrane prep-

arations to stimulate cytoplasmic glycolysis was

also decreased.

Pretreatment with nondiuretic mercurials at

equivalent levels did not result in a decreased

ability of membrane preparations to stimulate

glycolysis. The ATPase activity of the mem-

brane fragments was not significantly inhibited

until the mercurial dose was 8 mg Hg/kg.

Thus, a membrane enzyme system that can

be correlated with active Na�-K� transport has

been shown to be inhibited by mercurial diu-

retics in vitro and in vivo.

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A CELLULAR ACTION OF MERCURIAL DIURETICS’