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EFFECT OF ALDOSTERONE ON RENAL Na,K-ACTIVATED ADENOSINE-TRIPHOSPHATASE ACTIVITY IN NON-ADRENALECTOMIZED RATS
EFFECT
OF ALDOSTERONE
ON
RENAL
ADENOSINE-TRIPHOSPHATASE
Na,K-ACTIVATED ACTIVITY
NON-ADRENALECTOMIZED
IN
RATS
Fumitake IKEDA, Yasuo IKEDA, Chiyoko INAGAKI and Shuji TAKAORI Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Accepted September 11, 1978 It has been reported that the activity of Na, K-activated adenosinetriphosphatase (Na,K-ATPase) in the kidney is reduced by adrenalectomy and that the activity is restored by administration of aldosterone (1, 2). However, with the adrenalectomized rats, the restoration of the enzyme by aldosterone has been shown to take much longer time (i.e. 6-24 hr) as compared with the time (1-2 hr) for the maximal effect of aldosterone on the sodium reabsorption (3).
We attempted to determine whether or not aldosterone stimulates
Na,K-ATPase in correlation with the enhancement of Na reabsorption in non-adrenalec tomized rats. Male Wistar rats weighing 170-250 g were used.
Each animal was given water (3 ml
p.o.) and pitressin tannate in oil (0.5 U i.m), and then anesthetized 1 hr later with pen tobarbital sodium (30 mg/kg i.p.). Administration of 5 % mannitol through the femoral vein was started at a rate of 0.1 ml/ruin and continued during the experiment.
After a 30 min
equilibration period, one 30-min urine collection was made and then aldosterone (10 /tg per animal) was given i.v. to animals of the experimental group. The control animals were infused with 5 % mannitol through the experiment. The bladder was catheterized and sodium and potassium in the urine were determined by flame photometry and the chloride content by a chloridemeter
(Hitachi 205),
(Hiranuma, Chloride Counter CL-3).
To
determine the activities of Mg and Na,K-ATPase, animals were sacrificed and the kidney
was removed quickly.
The tissue was dissected into three sections (cortex, medulla and
papilla) and homogenized in ice-cold 0.25 M sucrose containing 25 mM imidazole-HC1 (pH 7.0) and 1 mM EDTA-Tris (pH 7.0). The homogenate was diluted adequately and passed through two layers of gauze.
Incubation for ATPase assay was carried out for 10 min at
37'C in the medium containing 50 mM Tris-HCi (pH 7.4), 3 mM MgC12, 100 mM NaCI, 10 mM KC1, 1 mM EDTA-Tris, 3 mM ATP-Na2 and an aliquot of the tissue homogenate with or without l mM ouabain. 10% trichloroacetic
Reactions were terminated by the addition of 1 mM of
acid and inorganic phosphate
released during the incubation was
determined colorimetrically by the method of Chen et al. (4).
Mg-ATPase or Na,K-ATPase
activity was calculated as pmoles Pi/mg protein/hr of ouabain insensitive or ouabain sensi tive ATPase activity.
Nonenzymic release of inorganic phosphate from ATP during the
incubation was negligible.
Protein concentration was determined by the method of Lowry
et al. (5), using crystalline bovine serum albumin as a standard. As shown in Fig. 1, in the control animals given 5 % mannitol, urine volume, excretion of sodium, potassium and chloride was not altered during three 30 min periods of the experi ments.
In the aldosterone-treated
However,
excretion
chloride decreased sium increased 30 min
after
and
of potas
in the second
administration
as compared
the control and
sodium
and excretion
significantly
period
aldosterone
of
animals, no change was observed in urine volume.
animals.
of
to increases
in
The activities of Mg
Na,K-ATPase
of the
renal
cortex,
medulla
and papilla
were measured
animals
at the end of the second 30 min
period after administration to the
experimental
Na,K-ATPase the
of aldosterone
animals
activity
increased
Thus,
animals,
with
in
while Mg with ad
of aldosterone. even in non-adrenalectomized
there
was
an
increase
activity with no
ATPase
activity,
Na,K-ATPase
cretion
signifi
compared
ATPase
tantly
of
activity was not altered
ministration
rats,
1).
cortex
animals
as
creases in the control ATPase
(Table
in the
aldosterone-treated
cantly
in all
with
effect this
change
in the
after
The
Na,K on
Mg
increase
activity occurred
of ions
aldosterone.
and
in
in
concomi
urinary
ex
administration
of
specific
activity
of
FIG. 1. Effect of aldosterone (10 pg/animal) on urine volume (UV) and urinary ex cretion of Na (UNaV), K (UKV) and Cl (U(,IV) ions in rats. 0 ------0 Control animals. S ----e Aldosterone treated animals. Aldosterone was given i.v. in 10 min after the first 30-min urine collection. y : P (Aldosterone treated vs . Control) ;0.05. Values are given as means f SE of 5 determinations.
TABLE 1. Activity treated rats
of Mg
or Na,
K-ATPase
in kidneys
of control
and aldosterone
Values are given as means-,' SE of 5 determinations. *: P (Aldosterone treated vs.. Control)<0.01. Activities of Ivlg and Na,K-ATPase were determined in all experi mental animals 60 min after administration of aldosterone (10 ii/animal).
Na,K-ATPase However,
was highest
as increase
in the medulla
in cortical
Na,K-ATPase
creases in the medulla
and papilla,
a greater
are other
results
extent than
and those reported
activity may be explained
factors
membrane
ATPase
variable
structure.
activity
or decreased
unknown
protein
secondary
regulates
The
the time course
been
of the enzyme
evident
tubular
renal Na,K-ATPase
suggest that,
the
in
of change
between
to our
in Na,K-ATPase
1) the rats used in our ex
used were given pitressin tannate antidiuretic
to minimize
(6), increased
transport
in
Na,K
to increased
synthesis
of an
of the enzyme
by aldosterone
in a physiological
activity in connection
changes
in renal
to be due
to
hormone.
artificial
by aldosterone
suggested
protein
sodium
than
by aldosterone
the enzyme (7) or increase of specific activity
of absolute
aldosterone.
Discrepancy
factors:
of increase has
given
may be regulated
with detergents
rats
in the present experiments
functional
more
in the level of intrinsic
mechanism
degradation
was
were
tubules.
2) the animals
change
was not treated
that unmasks
to stimulation
data obtained
with
of adrenalectomized
synthesis
of the renal
(1, 2) concerning
were non-adrenalectomized.
the
activity
by any one of the following
eliminate
preparation
the animals
the cortical tubules
sections
periments
3) ATPase
before
(8).
The
state, aldosterone
with a renal cation transport.
REFERENCES 1) JORGENSEN,P.L.: Regulation of the (Na+-{-K+)-activated ATP hydrolyzing enzyme system in rat kidney. 11. The effect of aldosterone on the activity in kidneys of adrenalectornized rats. Biochini. 13iophys. Acta 192, 326-334 (1969) 2) HENDLER,E.D., TORRETTI,J., KuroR, L. AND EPSTEIN,F.H.: Effects of adrenalectomy and hormone replacement on Na-K-ATPase in renal tissue. Arn. J. Physiol. 222, 754-760 (1972) 3) WIEDERHOLT,M., STOLTE,H., BRECHT,J.P. AND 1-IIERIIOLZER, K.: Mikropunktionsuntersuch ungen fiber den Einfluss von Aldosteron, Cortison and Dexamethason auf die renale Natriumresorption adrenalektomierter Ratten. Pffugcrs Arch. 292, 316-333 (1966) 4) CHEN,P.S. JR., TORIBARA,T.Y. AND WARNER,H.: Microdetermination of phosphorus. Analyt. Chem. 28, 1756-1758 (1956) 5) LOWRY, O.H., ROSEBROUGH,N.J., FARR, A.L. AND RANDALL,R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 6) KNOX, W.H. AND SEN, A.K.: Mechanism of action of aldosterone with particular reference to (Na, K)-ATPase. Ann. N. Y. Acad. Sci. 242, 471-488 (1974) 7) SCHMIDT,U., SCHMIDT,J., SCHMIDT,H. AND DUBACH, U.C.: Sodium and potassium
8)
activated ATPase. A possible target of aldosterone. J. clip. Invest. 55, 655-660 (1975) WESTENEELDER, C., AREVALO,G.J., BARANOWSKI,R.L., KURTZMAN,N.A. AND KATZ, A.1.: Relationship between mineralocoticoids and renal Na, K-ATPase; sodium reabsorp tion. Am. J. Physiol. 233, F593-599 (1977)
OF ALDOSTERONE
ON
RENAL
ADENOSINE-TRIPHOSPHATASE
Na,K-ACTIVATED ACTIVITY
NON-ADRENALECTOMIZED
IN
RATS
Fumitake IKEDA, Yasuo IKEDA, Chiyoko INAGAKI and Shuji TAKAORI Department of Pharmacology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Accepted September 11, 1978 It has been reported that the activity of Na, K-activated adenosinetriphosphatase (Na,K-ATPase) in the kidney is reduced by adrenalectomy and that the activity is restored by administration of aldosterone (1, 2). However, with the adrenalectomized rats, the restoration of the enzyme by aldosterone has been shown to take much longer time (i.e. 6-24 hr) as compared with the time (1-2 hr) for the maximal effect of aldosterone on the sodium reabsorption (3).
We attempted to determine whether or not aldosterone stimulates
Na,K-ATPase in correlation with the enhancement of Na reabsorption in non-adrenalec tomized rats. Male Wistar rats weighing 170-250 g were used.
Each animal was given water (3 ml
p.o.) and pitressin tannate in oil (0.5 U i.m), and then anesthetized 1 hr later with pen tobarbital sodium (30 mg/kg i.p.). Administration of 5 % mannitol through the femoral vein was started at a rate of 0.1 ml/ruin and continued during the experiment.
After a 30 min
equilibration period, one 30-min urine collection was made and then aldosterone (10 /tg per animal) was given i.v. to animals of the experimental group. The control animals were infused with 5 % mannitol through the experiment. The bladder was catheterized and sodium and potassium in the urine were determined by flame photometry and the chloride content by a chloridemeter
(Hitachi 205),
(Hiranuma, Chloride Counter CL-3).
To
determine the activities of Mg and Na,K-ATPase, animals were sacrificed and the kidney
was removed quickly.
The tissue was dissected into three sections (cortex, medulla and
papilla) and homogenized in ice-cold 0.25 M sucrose containing 25 mM imidazole-HC1 (pH 7.0) and 1 mM EDTA-Tris (pH 7.0). The homogenate was diluted adequately and passed through two layers of gauze.
Incubation for ATPase assay was carried out for 10 min at
37'C in the medium containing 50 mM Tris-HCi (pH 7.4), 3 mM MgC12, 100 mM NaCI, 10 mM KC1, 1 mM EDTA-Tris, 3 mM ATP-Na2 and an aliquot of the tissue homogenate with or without l mM ouabain. 10% trichloroacetic
Reactions were terminated by the addition of 1 mM of
acid and inorganic phosphate
released during the incubation was
determined colorimetrically by the method of Chen et al. (4).
Mg-ATPase or Na,K-ATPase
activity was calculated as pmoles Pi/mg protein/hr of ouabain insensitive or ouabain sensi tive ATPase activity.
Nonenzymic release of inorganic phosphate from ATP during the
incubation was negligible.
Protein concentration was determined by the method of Lowry
et al. (5), using crystalline bovine serum albumin as a standard. As shown in Fig. 1, in the control animals given 5 % mannitol, urine volume, excretion of sodium, potassium and chloride was not altered during three 30 min periods of the experi ments.
In the aldosterone-treated
However,
excretion
chloride decreased sium increased 30 min
after
and
of potas
in the second
administration
as compared
the control and
sodium
and excretion
significantly
period
aldosterone
of
animals, no change was observed in urine volume.
animals.
of
to increases
in
The activities of Mg
Na,K-ATPase
of the
renal
cortex,
medulla
and papilla
were measured
animals
at the end of the second 30 min
period after administration to the
experimental
Na,K-ATPase the
of aldosterone
animals
activity
increased
Thus,
animals,
with
in
while Mg with ad
of aldosterone. even in non-adrenalectomized
there
was
an
increase
activity with no
ATPase
activity,
Na,K-ATPase
cretion
signifi
compared
ATPase
tantly
of
activity was not altered
ministration
rats,
1).
cortex
animals
as
creases in the control ATPase
(Table
in the
aldosterone-treated
cantly
in all
with
effect this
change
in the
after
The
Na,K on
Mg
increase
activity occurred
of ions
aldosterone.
and
in
in
concomi
urinary
ex
administration
of
specific
activity
of
FIG. 1. Effect of aldosterone (10 pg/animal) on urine volume (UV) and urinary ex cretion of Na (UNaV), K (UKV) and Cl (U(,IV) ions in rats. 0 ------0 Control animals. S ----e Aldosterone treated animals. Aldosterone was given i.v. in 10 min after the first 30-min urine collection. y : P (Aldosterone treated vs . Control) ;0.05. Values are given as means f SE of 5 determinations.
TABLE 1. Activity treated rats
of Mg
or Na,
K-ATPase
in kidneys
of control
and aldosterone
Values are given as means-,' SE of 5 determinations. *: P (Aldosterone treated vs.. Control)<0.01. Activities of Ivlg and Na,K-ATPase were determined in all experi mental animals 60 min after administration of aldosterone (10 ii/animal).
Na,K-ATPase However,
was highest
as increase
in the medulla
in cortical
Na,K-ATPase
creases in the medulla
and papilla,
a greater
are other
results
extent than
and those reported
activity may be explained
factors
membrane
ATPase
variable
structure.
activity
or decreased
unknown
protein
secondary
regulates
The
the time course
been
of the enzyme
evident
tubular
renal Na,K-ATPase
suggest that,
the
in
of change
between
to our
in Na,K-ATPase
1) the rats used in our ex
used were given pitressin tannate antidiuretic
to minimize
(6), increased
transport
in
Na,K
to increased
synthesis
of an
of the enzyme
by aldosterone
in a physiological
activity in connection
changes
in renal
to be due
to
hormone.
artificial
by aldosterone
suggested
protein
sodium
than
by aldosterone
the enzyme (7) or increase of specific activity
of absolute
aldosterone.
Discrepancy
factors:
of increase has
given
may be regulated
with detergents
rats
in the present experiments
functional
more
in the level of intrinsic
mechanism
degradation
was
were
tubules.
2) the animals
change
was not treated
that unmasks
to stimulation
data obtained
with
of adrenalectomized
synthesis
of the renal
(1, 2) concerning
were non-adrenalectomized.
the
activity
by any one of the following
eliminate
preparation
the animals
the cortical tubules
sections
periments
3) ATPase
before
(8).
The
state, aldosterone
with a renal cation transport.
REFERENCES 1) JORGENSEN,P.L.: Regulation of the (Na+-{-K+)-activated ATP hydrolyzing enzyme system in rat kidney. 11. The effect of aldosterone on the activity in kidneys of adrenalectornized rats. Biochini. 13iophys. Acta 192, 326-334 (1969) 2) HENDLER,E.D., TORRETTI,J., KuroR, L. AND EPSTEIN,F.H.: Effects of adrenalectomy and hormone replacement on Na-K-ATPase in renal tissue. Arn. J. Physiol. 222, 754-760 (1972) 3) WIEDERHOLT,M., STOLTE,H., BRECHT,J.P. AND 1-IIERIIOLZER, K.: Mikropunktionsuntersuch ungen fiber den Einfluss von Aldosteron, Cortison and Dexamethason auf die renale Natriumresorption adrenalektomierter Ratten. Pffugcrs Arch. 292, 316-333 (1966) 4) CHEN,P.S. JR., TORIBARA,T.Y. AND WARNER,H.: Microdetermination of phosphorus. Analyt. Chem. 28, 1756-1758 (1956) 5) LOWRY, O.H., ROSEBROUGH,N.J., FARR, A.L. AND RANDALL,R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275 (1951) 6) KNOX, W.H. AND SEN, A.K.: Mechanism of action of aldosterone with particular reference to (Na, K)-ATPase. Ann. N. Y. Acad. Sci. 242, 471-488 (1974) 7) SCHMIDT,U., SCHMIDT,J., SCHMIDT,H. AND DUBACH, U.C.: Sodium and potassium
8)
activated ATPase. A possible target of aldosterone. J. clip. Invest. 55, 655-660 (1975) WESTENEELDER, C., AREVALO,G.J., BARANOWSKI,R.L., KURTZMAN,N.A. AND KATZ, A.1.: Relationship between mineralocoticoids and renal Na, K-ATPase; sodium reabsorp tion. Am. J. Physiol. 233, F593-599 (1977)