366

Biochimica et Biophysica Acta, 762 (1983) 366-371

Elsevier Biomedical Press

BBA 11151

ANDROGENIC STIMULATION OF ENDOCYTOSIS, AMINO ACID AND HEXOSE TRANSPORT

IN MOUSE KIDNEY CORTEX INVOLVES INCREASED CALCIUM FLUXES
A L F R E D D. GOLDSTONE

a,b

HAROLD KOENIG a and C H U N G Y. LU ~

Neurology Service, VA Lakeside Medical Center, 333 East Huron Street, Chicago, I L 60611 and Departments of Neurology and
Biochemistry, Northwestern University Medical School, Chicago, IL 60611 (U.S.A.)
(Received November l lth, 1982)

Key words: Testosterone stimulation," Endocytosis," Amino acid transport," Hexose transport," Ca 2 + flux; (Mouse kidney cortex)

Testosterone was previously shown to induce an early (less than 1 min) receptor-dependent stimulation of
endocytosis, hexose and amino acid transport in mouse kidney cortex (Koenig, H., Goldstone, A. and Lu,
C.Y. (1982) Biochem. Biophys. Res. Commun. 104, 165-172). Testosterone ( 1 0 - s M) has now been found
to stimulate rapidly (less than 30 s) the influx and efflux of 4SCa2+ in cortex slices. Testosterone also
decreased mitochondriai 4 5 C a and augmented soluble 4 5 C a , indicating a mobilization of intracellular calcium.

Incubation of cortex slices in calcium-free medium without or with 2.5 mM EGTA decreased basal
endocytosis, hexose and amino acid transport and blocked the hormonal response. 100 i~M verapamil blocked
the hormonal response without affecting basal transport. The calcium ionophore A23187 rapidly stimulated
endocytosis, hexose and amino acid transport. These data indicate that androgenic stimulation of membrane
transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular
calcium. Increased cytosolic Ca 2+ is probably the regulatory signal for these transport processes.

Introduction

The mouse kidney is a valuable model for investigating the mechanism of androgenic hormone
action as it exhibits a vigorous trophic response to
testosterone that is receptor-dependent [1,2] and is
characterized by marked organ hypertrophy, enhanced RNA and protein synthesis, and increases
in fl-glucuronidase and several other specific proteins [3]. We showed that the androgenic response
in mouse kidney features augmented activity of the
lysosomal system in proximal tubules, which is
expressed morphologically as a striking autophagy,
an accumulation of enlarged membrane-filled

Abbreviation: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

0167-4889/83/0000-0000/$03.00 1983 Elsevier Science Publishers

lysosomes (myeloid bodies) and exocytosis of these

lysosomes into the tubule lumen, and biochemically in increased kidney activities of numerous
lysosomal enzymes, a marked lysosomal enzymuria and proteinuria [4,5], and decreased enzyme latency and membrane stability of kidney
enzymes [6,7]. Testosterone also evokes changes in
proximal tubule mitochondria, reflected in alterations in size, fine structure [5] and equilibrium
density (unpublished data), an increase in the inner mitochondrial membrane enzyme, cytochrome
c oxidase [5], and enhanced cortical tissue respiration (unpublished data). Augmented ornithine decarboxylase activity and polyamine synthesis have
been shown to play an essential role in the mediation of the lysosomal and mitochondrial effects of
testosterone in mouse kidney proximal tubules,
and to be partially involved in mediating the
growth-promoting effect of the hormone [8].

367
Recently we found that physiological concentrations of testosterone induce a rapid (less
than 1 min) increase in endocytosis, hexose and
amino acid transport in mouse kidney cortex involving the proximal tubules [9]. This rapid response appears to be receptor-dependent as it
exhibits steroid specificity and does not occur in
the androgen-insensitive tfm/Y mouse, which lacks
androgen receptors [9]. It is generally thought that
steroid hormones regulate target cell function by
modulating RNA synthesis through the formation
of steroid-receptor complexes in the cell interior
[10]. However, the testosterone-induced increase in
RNA polymerase activity and chromatin template
activity appears later (about 15 min) [11] than the
stimulation of endocytosis, hexose and amino acid
transport (less than 1 min). Further, testosteroneinduced endocytosis is not initially dependent on
RNA or protein synthesis [12]. For these and other
reasons we concluded that the rapid testosterone
response represents a direct, receptor-mediated action of the steroid on the surface membrane of
target cells [9]. Since changes in plasma membrane
permeability to Ca 2 are a characteristic effect of
agents acting on surface membrane receptors
[13,14], we investigated the effects of testosterone
on Ca 2+ fluxes and distribution in mouse kidney
cortex slices. We report here experiments showing
that the testosterone-induced stimulation of endocytosis, hexose and amino acid transport in kidney
cortex involves an influx of extracellular calcium
and a mobilization of intracellular calcium.
Methods

2-Deoxy-D-[l-3H]glucose wa~ purchased from

Amersham International, Arlington Heights, IL.
a-[ 1-14C]Aminoisobutyric acid, 45Ca, and Aquasol
came from New England Nuclear, Boston, MA.
Horseradish peroxidase (Type II), ionophore
A23187 and testosterone (T 1500) were obtained
from Sigma Chemical Co., St. Louis, MO.
Verapamil (a-isopropyl-et-[(N-methyl-N-homoveratryl)-et-aminopropyl]-3,4-dimethoxyphenylacetonitrile hydrochloride) was a gift of Knoll Pharmaceutical Co., Whippany, NJ. Young adult female
A / J mice were bought from Jackson Labs., Bar
Harbor, ME, and C57-B1/6-COX mice from

Laboratory Supply Co., Indianapolis, IN. Animals

were killed by decapitation and the kidneys were
rapidly removed, decapsulated and placed in
chilled balanced salt solution containing (in mM):
122 NaC1, 5 KC1, 2.5 CaC12, 1.2 MgSO4, 2.6
Na2HPO 4, 0.6 K2HPO 4, 39 Hepes buffer, (pH

7.4).
Rates of endocytosis, hexose and amino acid
transport. As described previously [9], surface
cortical slices 300 /~m thick were prepared from
decapsulated kidneys and incubated in balanced
salt solution containing 6 mM glucose. 1 or 5
mg/ml horseradish peroxidase, 0.1 mM 4 #Ci/ml
a-[1-14C]aminoisobutyrate and 0.1 mM, 1/~Ci/ml
2-deoxy-D-[l-3H]glucose were added to the
medium to quantify endocytosis, amino acid and
hexose transport, respectively, Incubations were at
37C under 95% Ol/5% CO2 in a shaking incubator. Horseradish peroxidase uptake was typically
measured in 5 or 60 min incubations, and a-[114C]aminoisobutyrate and 2-deoxy-D-[1- 3H]glu_
cose uptake in 5 or 10 min incubations. The amount
of tracers taken up by slices during incubations at
0C was subtracted from 37C uptake to correct
for nonspecific uptake. Incubations were
terminated by placing tissue slices on ice, rinsing
three times in fresh medium and lysing in H20.
Protein [15], horseradish peroxidase [16], and radioactivity (by scintillation spectrometry) were assayed.
Rates of 45Ca2+ influx and efflux. For 45Ca
influx determination, cortex slices were preincubated in balanced salt solution with 6 mM glucose, transferred to fresh media containing 45Ca
(20 Ci/g calcium, 1.56 /~Ci/ml) without or with
l0 -8 M testosterone and incubated at 37C. Slices
were removed at various time intervals, washed
three times in unlabelled balanced salt solution,
and homogenized in 0.25 ml of balanced salt solution. Aliquots of each sample were counted in a
Beckman LS-250 liquid scintillation counter using
the t4C-window and assayed for protein. Corrections for nonspecific uptake of 45Ca were made by
subtracting the uptake at 0C. For 4~Ca efflux
determination, cortex slices were preincubated in
balanced salt solution with 6 mM glucose containing 5/~Ci/ml 45Ca for 20 min at 37C. The slices
were removed, washed six times with 10 ml of
unlabelled medium, and incubated in balanced

368

salt solution with 6 mM glucose at 37C in the

absence or presence of testosterone (10 -8 M). At
various time intervals 0.1 or 0.2 ml aliquots of the
medium were removed and 10 ml of Aquasol was
added to each aliquot in a vial for counting in a
liquid scintillation spectrometer. Correction for
losses of 4 5 C a from extracellular sites was made by
subtracting the efflux at 0C. At the end of the
incubations, slices were homogenized and assayed
for protein and 45Ca.
Subcellular distribution of 45Ca. To study the
effect of testosterone on the subcellular distribution of 45Ca in mouse kidney cortex, cortex slices
were preincubated in balanced salt solution and 6
mM glucose containing 3.5/~Ci/m145Ca 2+ at 37C
for 30 min. Slices were washed eight times at 0C
and incubated in fresh, nonradioactive medium
without and with 10 8 M testosterone for 10 rain
at 37C. Incubations were terminated by placing
slices in 2 ml of ice-cold 0.24 M sucrose/2 mM
K+-Hepes (pH 7.4) with 10 /~M Ruthenium red,
0.1 mM La203 and 1 mM EGTA to inhibit
artifactual redistribution of 45Ca, All subsequent
steps were at 4C. Slices were homogenized with
two strokes of a rapidly rotating motor-driven
pestle. Homogenates were centrifuged at 800 g
for 10 min, the crude nuclear pellet resuspended in
1.5 ml of medium and recentrifuged to deposit a
nuclear fraction. The combined supernatants were
centrifuged at 16000 g for 20 rain to pellet the
mitochondrial fraction, and the supernatant was
centrifuged at 105 000 g for 60 min to separate
the microsomal fraction and soluble fraction. With
this fractionation scheme, the mitochondrial fraction contained about 90% of the total glutamate
dehydrogenase (mitochondrial marker) and 15 % of
the glucose-6-phosphatase (microsomal marker);
the microsomal fraction contained about 60% of
the glucose-6-phosphatase and 2% of the glutamate dehydrogenase (unpublished data). Fractions
were assayed for 45 Ca and proteins.
Results

Table I shows the effect of EGTA and verapamil

on basal and testosterone-stimulated uptake of
horseradish peroxidase, aminoisobutyrate and deoxyglucose by mouse kidney cortex slices. Two
experiments of this kind were done with similar

TABLE I
EFFECT OF EGTA A N D VERAPAMIL ON BASAL A N D
TESTOSTERONE-STIMULATED
UPTAKE
OF
H O R S E R A D I S H P E R O X I D A S E , ~x-[1-14C]AMINOISOB U T Y R A T E A N D 2 - D E O X Y - D - [ I - 3 H ] G L U C O S E BY
MOUSE K I D N E Y CORTEX
Cortex slices were preincubated for 10 rain irt balanced salt
solution and 6 mM glucose at 37C with 2.5 mM EGTA (zero
Ca2+), 100 t~M verapamil, or no agent. At zero-time slices were
incubated in fresh preincubation medium containing horseradish peroxidase, a-[1-J4C]aminoisobutyrate, 2-deoxy-D-[13H]glucose and 1 0 - 8 M testosterone or no hormone. The results are the means _+S.E. (n = 3).
Treat-

Horseradish

o~-[ l - 14 C]-

ment

peroxidase
( n g / m g protein
per 60 min)

Aminoisobutyrate
(nmol/mg
protein
per 10 min)

2-Deoxyo-[ 1- 3H]glucose
(nmol/mg
protein
per 10 min)

Control
Testosterone
EGTA

45.9_+5.4

1.9_+0.1

4.8_+0.2

96,1 +_9.9 b
28,8+3.7 a

2.7 0.2 ~
1.3-+0.1 b

6.5 -+0A ~'

3.9_+0.1 a

48,9_+3.6

2.9_+0.3

4.6_+0.2

29.6_+1.9 b'c

1.2_+0A b.c

3.6_+0.4 c

49.6_+7.3 c

1.8_+0.1 ~

3.9_+0.5 ~

Veraparnil
Testosterone
+
EGTA
Testosterone
+
verapamil

P < 0.05 vs. control (Student's t-test).

b p < 0.01 vs. control (Student's t-test).
" P < 0.01 vs. testosterone (Student's t-test).

results. We previously presented evidence [9] that

the uptake of horseradish peroxidase by cortex
slices gives a valid measure of fluid volume endocytosis; the s o d i u m - d e p e n d e n t u p t a k e of
aminoisobutyrate reflects the initial rate of amino
acid transport via the A (alanine-preferring) system; and deoxyglucose uptake occurs through a
stereospecific, saturable transport system that
mediates D-glucose transport. In confirmation of
earlier results [9], 10 nM testosterone induced a
rapid (less than 5 min) stimulation of endocytosis
( + 110%), hexose (+36%) and amino acid transport (+43%). The calcium chelator EGTA (2.5

369

raM) in the absence of added Ca 2+ decreased the

basal rate of horseradish peroxidase, aminoisobutyrate and deoxyglucose uptake. EGTA also
abolished the testosterone-induced increase in
horseradish peroxidase, aminoisobutyrate and deoxyglucose uptake. Calcium-free medium without
chelator evoked a similar decrement in the basal
rate of horseradish peroxidase, aminoisobutyrate
and deoxyglucose uptake, but was slightly less
effective in blocking the hormonal increment (data
not shown). The calcium channel blocker verapamil (100/~M) in the presence of 2.5 mM Ca2 did
not affect basal uptake of horseradish peroxidase,
aminoisobutyrate and deoxyglucose but abrogated
the hormonal response.
Table II presents the effect of the calcium ionophore A23187 on horseradish peroxidase,
aminoisobutyrate and deoxyglucose uptake by
cortex slices. These data, which are representative
of two similar experiments, indicate that ionophore A23187 rapidly stimulated the uptake of
horseradish peroxidase, aminoisobutyrate and deoxyglucose by kidney cortex.
Fig. 1 shows the effect of testosterone on the
influx of 45Ca into kidney cortex slices. Three

TABLE II
EFFECT OF I O N O P H O R E A23187 ON UPTAKE OF
H O R S E R A D I S H P E R O X I D A S E , a-[1-t4C]AMINOISO B U T Y R A T E A N D 2-DEOXY-D-[1-3H]GLUCOSE BY
MOUSE K I D N E Y CORTEX
After a 10 min preincubation in balanced salt solution and 6
mM glucose without (control) and with 5 v M ionophore
A23187, slices were incubated in fresh preincubation medium
containing horseradish peroxidase, a-[l-laC]aminoisobutyrate
and 2-deoxy-D-[l-3H]glucose for uptake measurements. Results
are means S.E. (n = 3).
Treatment

Control
A23187
a P < 0.05.
b P < 0.01.

Horseradish
peroxidase
(ng/mg
protein
per 60 min)

or-[1-14C]Aminoisobutyrate
(nmol/mg
protein per
10 min)

2-Deoxy-D[ 1- 3H]glucose
(nmol/mg
protein per
10 min)

56 + 7.4
916.8 ~

5.9 0.4
7.25:0.07 ~

4.67 + 0.008
6.13 +0.026 b

1.25

/"

,ag
t~

.75

E
_~ . s o

t~

t9

E
c

.25

I
0

Time (minutes)
Fig. I. Effect of testosterone on 45Ca2+ influx in mouse kidney
cortex. Cortex slices were incubated in balanced salt solution
with 6 mM glucose and 45Ca 2 without (
) and with
( . . . . . . ) 1 0 - S M testosterone. Results are means S.E.
(n = 3).

experiments of this kind were done with similar

results. These influx data (37C) were corrected
for 'nonspecific uptake' of 4 5 C a , i.e., 4 5 C a bound

~ ' ;c
~

'~"

.75

.5

0
E
c

.25

II
tl 1 2

20

60

Time

(minutes)

Fig. 2. Effect of testosterone on 45 Ca 2 + efflux in mouse kidney

cortex. Cortex slices were preincubated in balanced salt solution with 6 mM glucose and 45Ca for 20 rnin at 37C. Slices
were rinsed and incubated in 45Ca-free medium without
(O
) and with ( O . . . . . . O) 1 0 - S M testosterone at
37C. Results are means + S.E. (n = 3).

370
T A B L E III
E F F E C T OF T E S T O S T E R O N E ON T H E S U B C E L L U L A R D I S T R I B U T I O N OF 45Ca IN M O U S E K I D N E Y CORTEX
Experimental details are given in Methods. Data are means S.E. (n = 3).
Constituent
Treatment

Homogenate

Nuclear

Mitochondrial

Microsomal

Soluble

% Recovery

20.0 +0.6
15.7 +0.5 b

11.4 0.2
12.1 0.6

60.5 0.8
65.0 0.2 b

105.6 5
101.53

29.8 _+1.7
34.4 _+1.6

15.9 +0.6
16.4 _+0.6

35.7 _+1
33.2 1.3

Percentage Distribution
45Ca
Control
Testosterone

100
100

8
_+3

Protein
Control
100
_+8
Testosterone
100 -+2
45Ca Content ( n m o l / m g protein)
Control
3.84_+ 0.07
Testosterone
3.27 _+0.07 b

8.1 0.4
7.2 +0.7
18.5 _+1
15.9 _+0.9
1.71 _+0.06
1.94 _+0.06

2.65 _+0.16
1.85 -+ 0.09 a

2.80 _+0.07
2.94_+ 0.20

98.71.2
99.21

6.62 _+0.11
7.84 _+0.09 h

a p < 0.05 (testosterone vs. control).

b p < 0.01 (testosterone vs. control).

to extracellular sites, by subtracting the uptake of

at 0C. The influx 4 5 C a in control slices was
linear for about 1 rain and then decreased, whereas
in the presence of 10 nM testosterone the influx
of 4~Ca was linear for about 2 min before decreasing. Testosterone stimulated 45Ca influx into kidney cortex. Increased influx of 4 5 C a w a s detected
at 0.5 min of incubation and persisted for at least
10 min.
The effect of testosterone on the efflux of 4 5 C a
from cortex slices is shown in Fig. 2. Three experiments of this kind were performed with similar
results. It can be seen that 10 nM testosterone
enhanced the efflux of 4 5 C a from kidney cortex
during incubations of 0.5-60 min in length.
Table III demonstrates the effect of testosterone
on the intracellular distribution of 4 5 C a in mouse
kidney cortex. These data, which are representative of three separate experiments, show that
incubation of 45 Ca-preloaded cortex slices with 10
nM testosterone for 10 min caused a significant
loss of 45Ca from the mitochondrial fraction and
an increase of 4 5 C a in the soluble fraction.

45Ca

Discussion
Our experiments demonstrate that basal endocytosis, hexose and amino acid transport in mouse
renal cortex are dependent on the presence of

Ca 2+ in the external medium. Furthermore, the

early testosterone-induced stimulation of endocytosis, hexose and amino acid transport is associated with an increased influx of 4 5 C a . Increased
Ca 2+ influx is a requirement for this hormonal
response as it was abolished by chelation of
extracellular calcium (with EGTA) or inhibition of
calcium transport (with verapamil). Additional
support for the involvement of calcium influx
comes from the observation that the calcium ionophore A23187 mimicked the early action of
testosterone in inducing a rapid stimulation of
endocytosis, hexose and amino acid transport in
kidney cortex. The early increase in 4 5 C a efflux
induced by testosterone is consistent with a rapid
mobilization of membrane-bound 4 5 C a from an
intracellular pool into the cytosol and its subsequent extrusion from the cell, possibly by a
plasmalemmal
Ca2+-ATPase.
That
the
mitochondria are one source of this Ca 2+ is indicated by the finding that testosterone decreased
the mitochondrial 45Ca and increased the cytosolic
45Ca.
Elevated cytoplasmic Ca e+ has been variously
implicated in the stimulation of endocytosis [17,
18], glucose [19,20] and amino acid transport [21].
The present experiments and earlier results [9]
indicate that the rates of endocytosis, hexose and
amino acid transport in renal cortical cells are

371
altered in a coordinate m a n n e r u n d e r a variety of
e x p e r i m e n t a l conditions. This f i n d i n g implies the
existence of a c o m m o n regulatory m e c h a n i s m for
these diverse m e m b r a n e transport processes. Our
d a t a show that increased calcium fluxes precede or
coincide with a n d r o g e n i c s t i m u l a t i o n of these
m e m b r a n e functions, a n d support the view that
the free cytoplasmic Ca 2+ c o n c e n t r a t i o n may serve
as a c o m m o n regulatory signal for their control.
Testosterone s t i m u l a t i o n p r o b a b l y raises cytosolic
C a 2 in proximal tubule cells, b o t h by e n h a n c i n g
Ca 2+ influx from the extracellular mileu, a n d b y
m o b i l i z i n g m e m b r a n e - b o u n d Ca 2 from intracellular stores, m a i n l y mitochondria. Changes in p l a s m a
m e m b r a n e p e r m e a b i l i t y to Ca 2 are a characteristic effect of agents acting on surface m e m b r a n e
receptors [13,14]. The present findings are therefore consistent with the view that testosterone
evokes the rapid m e m b r a n e response b y interacting with a n d r o g e n receptors located i n the
p l a s m a l e m m a [9].

Acknowledgements
This i n v e s t i g a t i o n was s u p p o r t e d by the
Veterans A d m i n i s t r a t i o n a n d grants from the National Institutes of Health (NS 14700, N S 19047
a n d H L 26835). We t h a n k Mr. David Wolf for
technical assistance a n d Mrs. T. Howell for the
secretarial work.

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