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Dihydrotestosterone and 3α-androstanediol dynamics in the normal, involuted, and hyperplastic canine prostate
DIHYDROTESTOSTERONE AND DIOL DYNAltiICS IN THE NORMAL, H-YPERPLASTIC CANINE PROSTATE G. McKercher,
S. Chevalier,
K.D. Roberta,
3a-ANDROSTANE INVOLUTED, AND
G. Bleau, and A. Chapdelaine
Departments of Medicine, Obstetrics and Gynecology and of Biochemistry, University of Montreal, Maisonneke-Rosemont Hospital Research Center, Endocrine Laboratory, 5415 1’Assomption Blvd, Montreal, Quebec, Canada HlT 2M4. Received August 11, 1986 Revised April 1, 1987 ABSTRACT
Perfusion of canine prostatic tissue with [I, Z3H] Sc+androstane3a,17B-diol and [4-14C] dihydrotestosterone and the measurement of the isotopic concentrations at the steady state were used to calculate the metabolic dynamics of these steroids by prostatic tissue obtained from normal, castrated or androgen-treated dogs. From the results it was concluded that: Entry of both (1) and no saturation was observed steroids into the tissue was similar, with increasing concentrations of these androgens in the perfusion medium. In contrast, the entry of both steroids was reduced when the (2) Dihydrotestos: perfusion buffer was replaced with diluted plasma. terone in prostatic tissue was present in a diffusible form, whereas (3) The conversion of 3%170-andros3a,l7&androstanediol was not. tanediol to dihydrotestosterone was always higher than the conversion thus, the oxidative of dihydrotestosterone to 3 4176 -androstanediol; (4) The entry and uptake of both androgens was pathway was favored. greatly reduced in those prostates excised from castrated dogs. (5) The uptake of dihydrotestosterone by normal prostatic tissue was 3-to However, 6-fold higher than the uptake of 3c( ,17B-androstanediol. concentrations of dihydrotestosterone were higher intratissular obtained by increasing the concentration of 3a ,17 D-androstanedjol perfused over that observed by increasing the concentration of dihydroIn the hyperplastic tissue, the entry, testosterone perfused. (6) uptake and metabolism of the two androgens were similar to those but their intratissular concentrations observed in the normal gland, were found to be higher.
INTRUMJCTION
Prostatic tems which ticular, the
tissues
regulate
tissue
of
18/l-2
animal
androgen
species
concentrations
(DHT) , an androgen
dihydrotestosterone
development
STEROIDS
from several
benign
prostatic
July-August
(I-7) which
hyperplasia 1986
possess
(3.3-72)
is (W-l)
enzyme sysand,
in
implicated
parin
(6-10). 33
McKercher
56
Indeed,
it
within
this
has
the
(11,12)
been
prostate
and that
disorder
the
et al
tissue
that,
favors
the
5%androstane-3cz,17
is
in of
is
and also
the
to
net
formation
ability on
of to
to
DHT
induce
increase
a
weight
basis,
than DHT to
induce
canine
Moreover,
in
metabolism
androgens
their
more effective
results
BPH, steroid
different
related
DHT (13,14).
@dial
growth
canine
potency
experimentally of
in
an increase
relative
content
prostatic
shown
a higher
prostatic
DHT content
(14). it
More recently, DPH, the the
prostatic
major
cells,
DHT concentration
enzymatic
activity
treatments
In order
to
during
important design
in
elucidate
perfusion
of
vitro
the diol,
of
and release tissue
of
of
the
were assessed.
is
(20-25). intracellular dynamics
two androgens
Malathi diol of
the
between
dynamics
by those
the
usually
of
involves
steroids
within
in the canine and Gurpide
encounmost
the experimental
This technique related
prostate
DHT and its
using
parameters
dynamics
epithelial
not modified
the pitfalls
and Welch (17).
metabolic
and that
BPH (12).
were investigated
androgen
manner the
the
(15,16)
prostatic
interplay
some of
in human and canine
elevated
canine
two metabolically
various
as the hunan prostate
In this
of
to study
a high conversion
in
the
incubations,
a mixture
has been used
further
by Gurpide
calculation
not
dehydrogenase,
and to eliminate
precursor,
proposed
that,
which are known to induce
and the androgens tered
is
measured
namely 3a-hydroxysteroid
in vivo
the
has been demonstrated
(25)
the
the
and allows tissue
(18-20)
and
as well
have reported
to DHT in human BPH. uptake,
by normal
metabolism,
retention
and hyperplastic
prostate
.4XDROGES
DYXAMICS
IN DOG PROSTATE
57
MATERIALSAH) t4ETlfiJDS Radioactjve steroids [4- ‘4C]-DHT (57.5mCi/mmol) and [ 1, 2-3H]-5%androstane-3cl, 17&diol (40 Ci/mmol) were purchased from New England Nuclear, Boston, MA, Their radiochemical purity was and purified by Celite chromatography. verified by crystallization of an appropriate aliquot with authentic they were dissolved in ethanol and stored at 4°C. Unlabeled carrier; carrier DHT, and diol and estradiol were purchased from (E2) Steraloids, Wilton, NH, and purified by crystallization from methanol: water prior to use. Prostatic tissue Prostates were obtained from normal, castrated or androgen-treated After excision, the gland was dogs, as described previously (12). immediately dissected and the external envelope and urethra were discarded. The prostate was then rinsed several times with cold saline and kept on ice until the perfusion procedure. The weights of the prostates were from 8 to 15 g for normal glands, from 2 to 6 g for involuted glands, and from 15 to 30 g for the hyperplastic glands. Perfusion technique The tissue was sliced with a Stadie-Riggs microtome into slices of approximately 0.5 mm. After weighing, the slices were rinsed in cold saline medium, inserted into the vials used for perfusion, which were immersed in a water bath at 37’C. Each vial was connected to a 30 mL syringe fitted to a pump (model 351, Sage Instrunents) which delivered the medium at a constant rate of 9 mL/h. The perfusion medium consisted of a mixture of the two radioactive steroids 3H-diol and j4C-DHT dissolved in 0.3 mL of ethanol to which was added either 30 mL of saline medium, Krebs-Ringer bicarbonate solution (KRB) or diluted homologous plasma (I:2 in KRB buffer). When necessary, varying amounts of unlabeled steroids were added to achieve steroid concentrations ranging from 0.05 to IO uM in the perfusion medium. The vials were saturated with 02:CO2 (95:5) during the perfusions which were of 90 to 120 min duration, and samples of the perfusates were collected at 30-min periods in tubes containing 5 mL of ethyl ether. In general, the results obtained from the last collection were used for the calculation of the parameters. At the end of the perfusion, the slices were removed, rinsed with ice-cold saline and immediately homogenized in an all-glass homogenizer. In certain experiments, 3 to 6 samples (0.5 g each) were prepared from the same prostate in order to perfuse the two steroids at different concentrations. Isolation and purification of radioactive DHT and diol To each samole. 300 uo each of DHT and diol were added to account for procedural losses duriig purification. Following the measurement of the volumes of the perfusion medium and of each tissue homogenate, the samples were extracted 3 times with IO mL of ethyl ether. After extraction, the separation of DHT from the diol was achieved by paper chromatography on Whatman paper in the system benzene:heptane:methanol: water (34:66:80:20) for 4 h, followed by acetylation of each steroid as
58
McKercher
et al
described previously (26) and thin-layer chromatography of DHT acetate and diol diacetate on alumina, using the system benzene:ethyl acetate After each purification step, an aliquot of each (2:l) for 2.5 h. sample was transferred to a counting vial, evaporated to dryness, and the radioactivity was assayed after the addition of Liquifluor, using a Packard liquid scintillation spectrometer (model 3375) with a dual label discriminator. Nonradioactive steroids were located using the Zimmermann reaction for DHT (15% potassium hydroxide, 2% m-dinitrobenzene) and phosphomolybdic acid for the dial. The recovery of each steroid was established by gas-liquid chromamodel 805; support:Chromosorb W; tography (Packard gas chromatograph, stationary phase: 1% SE-30, Chromatographic Specialties, Brockville, gas: nitrogen; flow rate: 60 cc/min; temperature of the Ont.; carrier Recoveries of the diol and DHT column, 235°C and pressure, 10 psi). Carrier steroids (5 mg) were were calculated from a standard curve. then added to each sample of DHT acetate or diol disc ta e which were recrystallized from methanol-water until a constant ‘Hjf4C ratio was Concentrations of radioactive steroids in the perfusion and achieved. tissue samples were expressed in dpm/mL, after correction for losses and assuming a tissue specific gravity of 1. Calculation and definition of terms The parameters that describe the dynamics of both steroids in the prostatic slices are illustrated in Fig. 1; they have been calculated using the method of Gurpide and Welch (17).
UPTAKE-d@1
Figure
1. Experimental design. defined in the text.
The symbols
used
in
this
figure
are
cwicentrations '%-DHT
'H-dioL idpm/mL1
and
of
perfused
cancentratians of 'ti and in the perb.Eate (dpm/mL)
f4~-diol,
concentrations af ?j and ‘f4C-dial in the tissue (dpm/g) or (dpm/ml_, assuming a specific gravity of 1 for the tissue
Fractian of dial enters the tissue
fraction released
uptake
dial
and
‘Wake
DHT
of dial unchanged
ix
U-IT
that
or DHT that is from the tissue
retention of dial nr DtiT by the tissue compared to the perFusion medium
fPactiar-3 rrf DW form& inta dial.
that
is
trans-
fr*action of dial. that farmed into DHT
is
tsans-
~~n~~ntr~t~an~ af the medium (ng/mL)
din1
and
DHT
in
concenksations of the tissue (ngig%
dioP
and
DHT
in
60
.McKercher et al
RESULTS
Achievement In
of the steady
order
superfusion, intervals
to
verify
portions of
ratios
those
parameters
after
describe
the dynamics
perfusates
Effect slices
proteins
upon the
perfusions
plasma which used for molar both
was diluted
steroids,
is
in saline
tions
into
obtained
were impaired
as well
into
KRB buffer. the
at
90
prostatic
homologous
The steroids
final
radioactive were
used
and for
PM).
perfusions
are presented
perfusions
It
was found reduced
It was also
performed that
the
free
evident
as the uptake
in the presence
were
were collected
steroids
at iso-
two steroids
same concentrations
three
and therefore the cell.
(the
to 0.18
medium only.
DHT and diol fusion
desired
of these
made with
of
vial
Therefore,
were made with
were added to obtain
from 0.05
The results
of
slices
two parts
these
that
entry
prostatic
with
the perfusions
concentrations
parison
of
during
as the
perfusion.
of
and tissues
of the perfusion.
Certain
as well
75 min of
the start
plasma
was achieved
concentrations
min or more after of
state
were removed from the perfusion
constancy
that
from the
steady
The steroid
reached
calculated
when the
of tissue
30 min.
topic
state
of
in Table with
canine steroid that
steroids
plasma
diluted
proteins
available
the tissular
the two androgens
of plasma proteins.
1, and com-
for
bind dif-
concentraby the tissue
B.
55 xi 53 %r 44 v
Perfusion
15 v 15 i? 25 Jii
with
with
A.
PerFusion
OF
CO~CENTRATIDN DIOL/DHT (ng/XJ7K7mL)
COMPARISON
canine
saline
plasma
medium
0.17 0.14
0.29 0.20
1.06
0.75
0.26
1.43
0.71
0.15
0.70
15
16
15
55
41
19
0.26
0.28
0.21
0.76
0.75
0.76
0.41
1.20
0.42
2.91
6.09
3.53
uptake
CY
-VS. CANINE
uptake
( Cdiol)t
IN SAL INE MEDIUM
DHT
0.60
CY
DILUTED
1
DIDL
OF THE PERFUS 'ION CF 3H-DIOL/14C-DHT
TABLE
PLASMA
38
77
36
164
189
102
tCDHT)t
62
UzKercher
Perfusions
et al
of normal prostate
The results
of perfusions
concentrations that
the
the
slices. (a)
of
diol
concentration
influence
both
perfused
of
entry
used
is
in Fig.
illustrated
amount of steroid in the perfusion DHT, the
slices
(mean
Therefore, the diol
diol
2,
was not
6 value
released
for
is
2 A. 2 B.
diol
over
340 nM) is
It was also
into
the
the
range
unsaturable. increase
evident did
from the
(Table
cell
and DHT, 0.04
not
prostatic the
of
steroid
This
finding
was observed
with increasing
observed
to be present
It is
the mechanism regulating
slices
by the tissue
2.
with different
100 ng/mL)
steroid
that
where a linear
retained
(15 to
either
the
50 to
DHT would appear
in the
concentrations 2) that,
after
entry
and 0.17
in a diffusible
contrary into
the
respectively). form,
whereas
not.
[diol]
Figure
into
(from
medium.
of
glands
in Table
steroid
therefore,
steroids
concentrations
to
of
fraction
of
of normal prostate
and DHT are presented
It would appear, entry
glands
in
madium
(Us/o/h)
[DHq
in
medium
(Ho/q/h)
Concentration of diol in tissue (pg/g tissue) as a function of the concentration of diol perfused (pg/g/h). Concentration of DHT in tissue (1;g/g tissue) as a function of the concentration of DHT perfused (vg/g/h).
AXDROGEN
DYNAMCS
IN DOG PROSTATE
63
McKercher
et al
Moreover,
the
64
always
near
in
tissue
the
diol
unity,
of
to predominate of
the diol
diol
DHT were
perfused,
trations.
However,
PDHT_diol,
higher
by increasing
the
increasing
near
to
since
very
intratissular
high
of
diol
can be observed greatly
of that
reduced
in
The interconversion are also
entry, tissular fusion,
slices
hyperplastic
values gland
obtained
are
In
is
by the appear,
contrast,
the
concentrations
of
DHT concen-
greater of
than
DHT are
over
that
the
obtained
observed
for
of both
in
the diol from
by
diol
and
both
one
DHT at steroids
than in the normal prostate.
3.
It
and DHT were
both
dogs.
androgens
from hyperplastic
with normal tissue if
cas-
castrated of
with slices
dogs,
Table
14 weeks with the diol)
However,
with
illustrated
concentrations
obtained
the
from normal
excised
obtained
and uptake. of
obtained
prostates
in vivo
from those
concentrations the
of
The results
metabolism
DHT to
would
intratissular
perfused
and uptake
and intratissular
(dog treated
significantly
experiments
the entry
decreased.
prostates fer
these
of
of DHT perfused.
Dynamics of diol and DHT in prostates trated dogs and dogs with BPH The results
unity.
concentrations
concentration
the concentration
reaction
Pdiol_DHT
the
was metabolized
factor
even when high
higher,
leading
that
slices.
remains
DHT was several-fold
of
the diol
The oxidative
was
DHT ( Pdiol-DHT)
to
to DHT and was influenced
in the tissue
the
diol
The conversion
DHT.
concentrations.
The uptake uptake
to
of al 1 of
that
converted
than that
steroid
therefore,
factor
indicating
was
was lower
perfused
conversion
did
dif-
with regard
considers the
not
the
end of
were
higher
to
intra-
the in
perthe
.83
25/2500
so/50 50/50
Prostate from castrated dogs
.20 .I3
.83
.75
25/25
2500/30
Normal + 3, 17 -dial (14 weeks)
.60 .65 .68
.71 .60 .7E
15/15 15/50 15/1ao
15/15 100/15 500/15
Normal prostate
.75 .79 .80
Cy
Normal prostate
25/25 2500/30 25/2500
(ng/mL)/(ng/mL)
CONCENTRATIONS IN PERFUSION MEDIUM diol/DHT
Normal prostate
TISSUE SAMPLE
.Ol .Ol
.Ol
.Ol
.D5
.04 .02 .02
.02 .03 .04
.02 .06 .02
p
3
OIOL
.21 -20
.90 0.21 0.19
1.45
1.21
1.32
.72
0.70 0.93 0.88
1.43 1.15 1.93
1.06 0.53 O.hl
.87
.7i 1.09 .85
.88 .76 .88
.89 .83 .83
'Stake
I? 12
3,189
3,328
59
'19 77 94
41 134 989
55 1,341 1,391
cCdiol)t
.I9 .I8
.82 .a0 .80
.76 .63 .73
.75 .80 ‘00
.76 .84 .84
cx
1.22 1.00 1.08 0.48 0.91
l03 .02
1.35 0.94 1.00
1.09 1.24 1.01
1.10 1.14 1.08
Pdiol-DHT
.I9 .I2 .I7
.I6 .20 .21
.I2 .08 .ll
.I4 .I0 .I6
P
DHT
FACTORS, UPTAKE, INTRATISSUE CONCENTRATIONS OF TISSUE FROM NORMAL, CASTRATED OR TREATED DDGS
PDHT-diol
ENTRY, RELEASE, CONVERSION DIDL AND DHT BY PROSTATIC
TABLE
0.56 0.51
3.79 2.88 3.34
3.53 3.91 3.51
3.09 3.86 3.95
2.91 1.28 2.88
uptake
44 45
200 7,510 8,443
102 252 403
1,997
189 419
164 3,400 7,581
($HT)~
66
McKercher
The 21 h-l, value
et al
intracellular results
of
6.3
clearance fusions
not h-l)
of
(IC)
clearance shown) within
was always a given
DHT and diol
did
the
greater
tissue
not
were made with different
of
diol
(mean
than
that
to
of
be significant
steroid
concentrations.
on
application
of
DHT (mean
Variations
sample.
appear
value
in
the
when per-
DI!XUSSIDN The present tracer
study
superfusion
trations
of
is
method described
radioactive
steroids
measured at the isotopic from the ments
start
of
by Giorgi
The data
allow
in “batch”
trations
are
cellular would
that,
to
have shown that gen entry DHT are that
are
siologic
of
that little
conditions.
within
cannot
constants
of
higher
than those
consequence,
and the
extent
since
prostate of
were 90 min experi(25).
be estimated
interconversion,
protein
This binding
the hormones available
steroid
concen-
and lead
present Giorgi
to intra-
endogenously and co-workers
the regulation
conversion
concentrations
(18,21,22).
and the
the amounts of
that
in dog plasma
are higher
same at physiological
enzymes
Concen-
and Gurpide
the superfused
found
in the normal canine
ZOO-fold
metabolizing excess
be of
vitro
of hormones.
than those
and uptake
the
rate
in our experiments,
higher
in
shown in similar
and Malathi
parameters
such as the
concentrations appear
as already
of
the
and the superfusate
which was achieved
(18,Zl)
calculation
and the release
The fact
state,
and co-workers
of
by Gurpide and Welch (17).
the superfusion,
the
the
in the tissue
steady
incubations,
the entry
based
of
within
andro-
testosterone
as at would
of
concentrations
indicate the
to
that
cells
to the cells
are
the in
under phy-
Ah-DROGEN
The plasma canine
protein
plasma is
tracers
is
binding
used instead
greatly
This
metrium. albumin tein
It
steroid
is
laries;
however,
those
effect
is
as well
the
has
been
tissues
with
testosterone
and diffuse
into
The entry was similar
for
found
regulating of
of
both
steroids,
the
simple
of
as
tracers
binding
fraction
through
and
Moses
steroids
locally
into
to pro-
of the
the
(24)
such
capilin
that,
as the
from the binding
the steroids
found
by
illustrated
by Malathi
into
Giorgi
and
hunan
protein
of
our values
more unlikely,
with
(25)
were higher
in
than
of the mechanism
the slices
over
co-workers
for
by the
DHT and
slices
and Gurpide
a wide range testosterone,
tissue
linear
diol
in the medium, is compatible
or,
prostatic
and human prostatic
graphically
concentrations
concentrations
normal canine
however,
and DHT with canine
result,
diffusion,
of
the
free
There was no saturation
authors.
intratissular
steroid
of
passage
by
in human endo-
androgen
the
Giorgi
as found
experiments;
the entry
This
by
uptake
DHT and the diol
concentrations,
22).
a high
binding
only
of the
have been obtained
the canine
during
shown
the entry
the cell.
by these
androstenedione
blood
may dissociate
human BPH superfusion those
as to
67
when diluted
of estrogens
the
assumed that
from
it
results
due to
that,
perfusion,
the perfusion
generally
removed
target
prostate,
is
for
Similar
likely
IN DOG PROSTATE
demonstrate
of saline
during
and transcortin
(27).
studies
diminished.
Gurpide and Welch (17)
DYNAMICS
with
(18,21,
increase
of
increasing
with a mechanism of
a specific
carrier
system
of
high capacity. When the perfusions the entry
and uptake
of
were made with prostate both
the
diol
from a castrated
and DHT were greatly
reduced.
dog, It
McKercher
68
et al
has been demonstrated greatly
diminished
increase
in
the
tissue
from
effect
upon the
centration
that
following relative
of
This histological
steroids
into
can
exceeded
that
of
DHT but not
of
the
in
is
because
elements
in
receptors the
equal
are of
has
that
a large con-
perfused
affect
an
prostatic
intratissular
to
might also
for
binding
the
tissue
that
are
(50
the entry
4 to
of
(25)
diol
the
rarely
have also
shown
DHT but not the diol, tissue
gradient
Since the
have
to
medium
the entry
ability
must depend on specific to exist
while
(18,19,21,23)
from tissue
and unsaturable,
has been reported
that
8 times
of
and androstenedione.
similar
indicate
concentration
human and canine
DHT preferentially
that
steroids
and Gurpide
concentration
is
both
of
levels
Malathi
testosterone
to concentrate
uptake
can concentrate
using
two androgens
cellular
rig/g))
DHT at
slices
a positive
for
the
the medium.
and co-workers
tissue
for
while
human prostatic
demonstrated
partly
stromal
The decrease
(45
concentrate
concentration
Giorgi
of
modification
obtained
perfused
that
(28,29),
receptors
the tissue.
The values tissue
androgen
DHT,and consequently
DHT measured
ng/mL).
the
castration
dog.
uptake
and cytosolic
proportion
a castrated
of
nuclear
in the canine
of
the
intraprostate
(30,31). The a value
is
the fraction
unchanged
from the
tissue.
the canine
prostate
(IS),
Gurpide
(25)
measurable present (17).
for
steroid
by Giorgi
and by Grant and Giorgi DHT is
the human prostate,
a diffusible
On the contrary,
superfused
As reported
amounts (mean a value
in both
of
of
0.17),
is
released
and co-workers (23)
released indicating
and a bound form,
and in accordance
that
for
and Malathi
and
from the tissue
in
that
it
must be
which are in equilibrium
with the results
obtained
by
AXDROGEN
Malathi
and
@$0.06),
Gurpide
probably
because
In our superfusions study
of
Malathi
interconversion over
the
vitro
could
the
more efficient
of
an important
In the for
DHT (6.3
of
This
result,
which
(25)
can
explained
other less result plastic
be
binding steroid in
proteins being
a lower
tissue
is
in
of BPH in viva
(14,33).
(mean of
obtained
of diol
is
lower
could
of
this
be
con-
steroid
is
(14). with
lower
normal
than the with
prostatic IC for
that
strong
binding
in
canine
prostate,
the
15.2
and
why the diol
DHT, the diol
concentration
by the
clearance.
is
explain
accordance
available
canine
DHT under normal physiologic
performed
was always
reaction,
important
also
intracellular
of testosterone
h-l)
more
of
show a
in the
DHT concentration
precursor
with -in
also
Thus,
the
preferred
the reverse
the normal plasma concentration another
in
obtained cells
(12,32).
and could
intraprostatic
perfusions
tissue,
results
quantitatively
the induction
the
than that
is
medium
as in the
pathway is
over
intratissular
testosterone, source
as well
epithelial
coenzymes
of the diol
even if
since
ditions, higher
for
tissue,
diol--,DHT
the
69
retention.
laboratory,
pathway
into
human hyperplastic
prostatic
added
the perfusion
than that
prostatic
released
and DHT, the oxidative
why a higher
Moreover,
canine with
IN DOG PROSTATE
not
intracellular
canine
of
is
its
In our
oxidative
explain
diol
of the reaction
absence
the
following is
one. with
net predominance
prostate,
of
of the diol
reductive
in
of
and Gurpide
incubations
even
the
(25),
DYNAMICS
for
metabolism
The IC of h-l)
of
to
the
diol
Malathi DHT to
which
the
IC
(21 h-l). and Gurpide
receptors
would
and release,
the diol
appears
of
tissue,
or
result
in
and thereby
measured in the hyper-
be lower
than that
found
70
in
McKercher
et al
the normal prostate
diol
by
the
steroids
tissue.
were also
by in vivo
and could High
observed
treatment
and DHT in prostates
obtained
from dogs
with
metabolism
canine
androstenedione
androgen
(21).
These differences
to
higher
content
compared
also
induced
diol
(33,34) of
Further
obtained
of
of
the diol
with
the
of
to
while
the
diol
of
and
that
the
fibromuscular studies
the
of
androgen
thelial
cell
types
present
experimental
tissue
approach
is
reduced
by slices
of
by be
at
higher
and the
IC de-
might be related in
results cell
hunan 8PH is
with estrain
both
metsplasia
the relative cells
the
known that
combined
prostatic
receptors
reported could
well
as basal
and stromal
hyperplastic
found
estrogen
to elucidate
and estrogen been
of
as well
glandular
has
It
similar
testosterone,
the
increased
with the diol
presence
androgens
are
elements
dog.
are necessary of
in
with the human tissue
fibro-muscular
that
with
contrary,
is
prostate,
and DHT,do not dif-
perfused
uptake
canine
have obtained
and metabolism
in the dog by treatment
participation
interconversion
slices
entry
of
of the diol
On the
creases
tribution
two
with those
in vivo
and co-workers
prostatic
concentrations,
the
agree
in the hyperplastic
Giorgi
steroid
increase
the
tissue
concentrations
treated
and release
and DHT (18).
human prostate,
prostate
of
hyperplastic
These results
measured
from the normal gland.
the
concentrations
who measured high
parameters
the entry,
results
uptake of the
BPH.
The other
fer
intratissue
with the diol. (14)
i.e.,
from the higher
in the canine
Moore and co-workers
to induce
result
in
the
(35).
extent uptake
tissues.
useful
laboratory for
the
of and
The dis-
in the two prostatic our
an
epi-
(36). study
The of
the
AMJROGEX
metabolic as
under
behavior normal
of the and
androgens
pathologic
DYl’iAMICS
in each
conditions
prostatic and
71
IN DOG PROSTATE
during
cell
type
as well
hormonal
treat-
ment.
ACKNOULUNXNTS The authors are indebted to Claudette Boudreault and Monique Boire for their excellent secretarial assistance. Financial support of the medical Research Council of Canada and the FCAR, A.rebec,is gratefully acknowledged.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11. 12.
Nozu, K. and Tamaoki, B., ACTA ENDOCRINOL. (Kbh) 76, 608 (1974). J.D., BIOCHEMISTRY 14, 810 Taurog, J.D., Moore, R.J. and Wilson, (1975). Harper, M.E., Pierrepoint, C.G., Fahmy, A.R. and Griffiths, K., J. ENDOCRINOL. ,9, 213 (1971). J.D., J. STEROID BIOCHEM. Jacobi, J.H., Moore, R.J. and William, 8, 719 (1977). Hudson, R.W., J. STEROID BIOCHEM. 1-4, 579 (1981). Cowan, R.A., Cook, B., Cowan, S-K., Grant, J.K., Sirett, D.A.N. and Wallace, A.M., J. STEROID BIOCHEM. II_, 609 (1979). Krieg, M., Klijtzl, G., Kaufmann, J. and Voigt, K.D., ACTA ENDOCRINOL. (Kbh) 96, 422 (1981). Siiteri, P.K. and alson, J.D., J. CLIN. INVEST. ,9, 1737 (1970). Gloyna, R.E., Siiteri, P.K. and Wilson, J.D., J. CLIN. INVEST. 9, 1746 (1970). Geller, J., Albert, J., Lopez, D., Geller, S. and Niwayama, G., J. CLIN. ENDOCRINOL. METAB. 43, 686 (1976). 108, 445 (1981). Isaacs, J.T. and Coffey, D.S., ENDOCRINOLOGY Chapdelaine, A., Boulanger, P., Dupuy, G.M., Chevalier, S., Bieau, G. and Roberts, K.D., 62nd annual meeting of the Endocrine
15.
Society, Washington,D.C. (Abstract 6181 (1980). Isaacs, J.T., J. STEROID BIOCHEM. E, 749 (1983). Moore, R.J., Gazak, J-M., Quebbeman, J.F. and Wilson, J.D., J. CLIN. INVEST. 64, 1003 (1979). Walsh, P.C., Hutcmns, G.M. and Ewing, L.L., J. CLIN. INVEST.
16.
1772 (1983). Ewina. L.L..
13. 14.
17. 18. 19. 20.
72,
S.J. and Hiqoinbottom, E.G., ENDOCRINOLOGY Berry. __ l&2004 (;983). .’ Gurpide, E. and Welch, M., 3. BIOL. CHEM. Is, 5159 (1969). Giorgi, E.P., Grant, J.K., Stewart, J.C. and Reid, J., J. ENDOCRINOL.55, 421 (1972). Giorgi, E.P., J. ENDOCRINOL. ;s, 109 (1976). Giorgi, E.P., Moses, T.F., Grant, J.K., Scott, R. and Sine la ir, J., MOL.
CELL.ENDOCRINOL.
'&
271
(1974).
72
McKercher
21.
Giorgi, E.P., Stewart, J.C., Grant, J-K. and Scott, R., EIOCHEM. J. 123, 41 (1971). Giox, E.P., Shirley, I.M., Grant, J.K. and Stewart, J.C., BIOCHEM, J. 132, 465 (1973). Grant, J.K. and Giorgi, E.P., J. STERDID BIOC~EM. 2, 949 (1974). Giorgi, E.P. and Moses, T.F., 3. ENDOCRIND~. 65, 279 (1975). Malathi, K. and Gurpide, E., J. STEROID BIOCHEM. 8, 141 (1977). A., K.D., Bleau, G. and Chapdelaine, Dupuy, G.M., Roberts, J. STEROID BIOCHEM. 8, 1145 (1977). Suzuki, Y., Okumura, Y. and Sinohara, S., J. BIOCHEM. 85, 1195 (1979). Trachtenberg, J., Hicks, L.L. and Walsh, P.C., J. CLIN. INVEST. 65,1051 (19BO). Dub&, J.Y., FrBnette, G. and Tremblay, R.R., INVEST. UROL. E, 418 (19811. Dube, J.Y., Tremblay, R.R., Dionne, F.T. and Chapdelaine, P., J. STEROID BIOCHEM. IO, 449 (1979). Valotaire, Y., Chaisiri, N., Evans, B.A.J. and Pierrepoint, C.G., 3. ENDO~RINOL. 8l_, 147P (1979). McKerchet-, G., Chevalier, S., Roberts, K.D., Eleau, G. and Chapdelaine, A., J. STEROIU BIOCHEM. 2, 549 (1984). J. CLIN. INVEST, 7, 1093 (1976). Walsh, P.C. and Wilson, J.D., DeKlerk, D.P., Coffey, D.S., Ewing, L.C., McDermott, I.R., Reiner, C.H., Scott, W.W., Standberg, J.D., Talalay, P., W.G., Robinson, Walsh, P.C., Wheaton, F.G. and Zirkin, B.R., 3. CLIN. INVEST. 64, 842 (1979). and Neumann, F., ACTA Tunn, U., Senge, Th., Schenck, B. ENDOCRINOL. z, 373 (1979). Lamarre, D., Chevalier, S., McKercher, G., Bleau, G., Roberts, K.D. and Chapdelaine, A., J. STEROID BIOCHEM. 2, 1 (1985).
22. 23. 24. 25. 26. 27. 28.
29. 30. 31, 32. 33. 34.
35. 36.
et al
S. Chevalier,
K.D. Roberta,
3a-ANDROSTANE INVOLUTED, AND
G. Bleau, and A. Chapdelaine
Departments of Medicine, Obstetrics and Gynecology and of Biochemistry, University of Montreal, Maisonneke-Rosemont Hospital Research Center, Endocrine Laboratory, 5415 1’Assomption Blvd, Montreal, Quebec, Canada HlT 2M4. Received August 11, 1986 Revised April 1, 1987 ABSTRACT
Perfusion of canine prostatic tissue with [I, Z3H] Sc+androstane3a,17B-diol and [4-14C] dihydrotestosterone and the measurement of the isotopic concentrations at the steady state were used to calculate the metabolic dynamics of these steroids by prostatic tissue obtained from normal, castrated or androgen-treated dogs. From the results it was concluded that: Entry of both (1) and no saturation was observed steroids into the tissue was similar, with increasing concentrations of these androgens in the perfusion medium. In contrast, the entry of both steroids was reduced when the (2) Dihydrotestos: perfusion buffer was replaced with diluted plasma. terone in prostatic tissue was present in a diffusible form, whereas (3) The conversion of 3%170-andros3a,l7&androstanediol was not. tanediol to dihydrotestosterone was always higher than the conversion thus, the oxidative of dihydrotestosterone to 3 4176 -androstanediol; (4) The entry and uptake of both androgens was pathway was favored. greatly reduced in those prostates excised from castrated dogs. (5) The uptake of dihydrotestosterone by normal prostatic tissue was 3-to However, 6-fold higher than the uptake of 3c( ,17B-androstanediol. concentrations of dihydrotestosterone were higher intratissular obtained by increasing the concentration of 3a ,17 D-androstanedjol perfused over that observed by increasing the concentration of dihydroIn the hyperplastic tissue, the entry, testosterone perfused. (6) uptake and metabolism of the two androgens were similar to those but their intratissular concentrations observed in the normal gland, were found to be higher.
INTRUMJCTION
Prostatic tems which ticular, the
tissues
regulate
tissue
of
18/l-2
animal
androgen
species
concentrations
(DHT) , an androgen
dihydrotestosterone
development
STEROIDS
from several
benign
prostatic
July-August
(I-7) which
hyperplasia 1986
possess
(3.3-72)
is (W-l)
enzyme sysand,
in
implicated
parin
(6-10). 33
McKercher
56
Indeed,
it
within
this
has
the
(11,12)
been
prostate
and that
disorder
the
et al
tissue
that,
favors
the
5%androstane-3cz,17
is
in of
is
and also
the
to
net
formation
ability on
of to
to
DHT
induce
increase
a
weight
basis,
than DHT to
induce
canine
Moreover,
in
metabolism
androgens
their
more effective
results
BPH, steroid
different
related
DHT (13,14).
@dial
growth
canine
potency
experimentally of
in
an increase
relative
content
prostatic
shown
a higher
prostatic
DHT content
(14). it
More recently, DPH, the the
prostatic
major
cells,
DHT concentration
enzymatic
activity
treatments
In order
to
during
important design
in
elucidate
perfusion
of
vitro
the diol,
of
and release tissue
of
of
the
were assessed.
is
(20-25). intracellular dynamics
two androgens
Malathi diol of
the
between
dynamics
by those
the
usually
of
involves
steroids
within
in the canine and Gurpide
encounmost
the experimental
This technique related
prostate
DHT and its
using
parameters
dynamics
epithelial
not modified
the pitfalls
and Welch (17).
metabolic
and that
BPH (12).
were investigated
androgen
manner the
the
(15,16)
prostatic
interplay
some of
in human and canine
elevated
canine
two metabolically
various
as the hunan prostate
In this
of
to study
a high conversion
in
the
incubations,
a mixture
has been used
further
by Gurpide
calculation
not
dehydrogenase,
and to eliminate
precursor,
proposed
that,
which are known to induce
and the androgens tered
is
measured
namely 3a-hydroxysteroid
in vivo
the
has been demonstrated
(25)
the
the
and allows tissue
(18-20)
and
as well
have reported
to DHT in human BPH. uptake,
by normal
metabolism,
retention
and hyperplastic
prostate
.4XDROGES
DYXAMICS
IN DOG PROSTATE
57
MATERIALSAH) t4ETlfiJDS Radioactjve steroids [4- ‘4C]-DHT (57.5mCi/mmol) and [ 1, 2-3H]-5%androstane-3cl, 17&diol (40 Ci/mmol) were purchased from New England Nuclear, Boston, MA, Their radiochemical purity was and purified by Celite chromatography. verified by crystallization of an appropriate aliquot with authentic they were dissolved in ethanol and stored at 4°C. Unlabeled carrier; carrier DHT, and diol and estradiol were purchased from (E2) Steraloids, Wilton, NH, and purified by crystallization from methanol: water prior to use. Prostatic tissue Prostates were obtained from normal, castrated or androgen-treated After excision, the gland was dogs, as described previously (12). immediately dissected and the external envelope and urethra were discarded. The prostate was then rinsed several times with cold saline and kept on ice until the perfusion procedure. The weights of the prostates were from 8 to 15 g for normal glands, from 2 to 6 g for involuted glands, and from 15 to 30 g for the hyperplastic glands. Perfusion technique The tissue was sliced with a Stadie-Riggs microtome into slices of approximately 0.5 mm. After weighing, the slices were rinsed in cold saline medium, inserted into the vials used for perfusion, which were immersed in a water bath at 37’C. Each vial was connected to a 30 mL syringe fitted to a pump (model 351, Sage Instrunents) which delivered the medium at a constant rate of 9 mL/h. The perfusion medium consisted of a mixture of the two radioactive steroids 3H-diol and j4C-DHT dissolved in 0.3 mL of ethanol to which was added either 30 mL of saline medium, Krebs-Ringer bicarbonate solution (KRB) or diluted homologous plasma (I:2 in KRB buffer). When necessary, varying amounts of unlabeled steroids were added to achieve steroid concentrations ranging from 0.05 to IO uM in the perfusion medium. The vials were saturated with 02:CO2 (95:5) during the perfusions which were of 90 to 120 min duration, and samples of the perfusates were collected at 30-min periods in tubes containing 5 mL of ethyl ether. In general, the results obtained from the last collection were used for the calculation of the parameters. At the end of the perfusion, the slices were removed, rinsed with ice-cold saline and immediately homogenized in an all-glass homogenizer. In certain experiments, 3 to 6 samples (0.5 g each) were prepared from the same prostate in order to perfuse the two steroids at different concentrations. Isolation and purification of radioactive DHT and diol To each samole. 300 uo each of DHT and diol were added to account for procedural losses duriig purification. Following the measurement of the volumes of the perfusion medium and of each tissue homogenate, the samples were extracted 3 times with IO mL of ethyl ether. After extraction, the separation of DHT from the diol was achieved by paper chromatography on Whatman paper in the system benzene:heptane:methanol: water (34:66:80:20) for 4 h, followed by acetylation of each steroid as
58
McKercher
et al
described previously (26) and thin-layer chromatography of DHT acetate and diol diacetate on alumina, using the system benzene:ethyl acetate After each purification step, an aliquot of each (2:l) for 2.5 h. sample was transferred to a counting vial, evaporated to dryness, and the radioactivity was assayed after the addition of Liquifluor, using a Packard liquid scintillation spectrometer (model 3375) with a dual label discriminator. Nonradioactive steroids were located using the Zimmermann reaction for DHT (15% potassium hydroxide, 2% m-dinitrobenzene) and phosphomolybdic acid for the dial. The recovery of each steroid was established by gas-liquid chromamodel 805; support:Chromosorb W; tography (Packard gas chromatograph, stationary phase: 1% SE-30, Chromatographic Specialties, Brockville, gas: nitrogen; flow rate: 60 cc/min; temperature of the Ont.; carrier Recoveries of the diol and DHT column, 235°C and pressure, 10 psi). Carrier steroids (5 mg) were were calculated from a standard curve. then added to each sample of DHT acetate or diol disc ta e which were recrystallized from methanol-water until a constant ‘Hjf4C ratio was Concentrations of radioactive steroids in the perfusion and achieved. tissue samples were expressed in dpm/mL, after correction for losses and assuming a tissue specific gravity of 1. Calculation and definition of terms The parameters that describe the dynamics of both steroids in the prostatic slices are illustrated in Fig. 1; they have been calculated using the method of Gurpide and Welch (17).
UPTAKE-d@1
Figure
1. Experimental design. defined in the text.
The symbols
used
in
this
figure
are
cwicentrations '%-DHT
'H-dioL idpm/mL1
and
of
perfused
cancentratians of 'ti and in the perb.Eate (dpm/mL)
f4~-diol,
concentrations af ?j and ‘f4C-dial in the tissue (dpm/g) or (dpm/ml_, assuming a specific gravity of 1 for the tissue
Fractian of dial enters the tissue
fraction released
uptake
dial
and
‘Wake
DHT
of dial unchanged
ix
U-IT
that
or DHT that is from the tissue
retention of dial nr DtiT by the tissue compared to the perFusion medium
fPactiar-3 rrf DW form& inta dial.
that
is
trans-
fr*action of dial. that farmed into DHT
is
tsans-
~~n~~ntr~t~an~ af the medium (ng/mL)
din1
and
DHT
in
concenksations of the tissue (ngig%
dioP
and
DHT
in
60
.McKercher et al
RESULTS
Achievement In
of the steady
order
superfusion, intervals
to
verify
portions of
ratios
those
parameters
after
describe
the dynamics
perfusates
Effect slices
proteins
upon the
perfusions
plasma which used for molar both
was diluted
steroids,
is
in saline
tions
into
obtained
were impaired
as well
into
KRB buffer. the
at
90
prostatic
homologous
The steroids
final
radioactive were
used
and for
PM).
perfusions
are presented
perfusions
It
was found reduced
It was also
performed that
the
free
evident
as the uptake
in the presence
were
were collected
steroids
at iso-
two steroids
same concentrations
three
and therefore the cell.
(the
to 0.18
medium only.
DHT and diol fusion
desired
of these
made with
of
vial
Therefore,
were made with
were added to obtain
from 0.05
The results
of
slices
two parts
these
that
entry
prostatic
with
the perfusions
concentrations
parison
of
during
as the
perfusion.
of
and tissues
of the perfusion.
Certain
as well
75 min of
the start
plasma
was achieved
concentrations
min or more after of
state
were removed from the perfusion
constancy
that
from the
steady
The steroid
reached
calculated
when the
of tissue
30 min.
topic
state
of
in Table with
canine steroid that
steroids
plasma
diluted
proteins
available
the tissular
the two androgens
of plasma proteins.
1, and com-
for
bind dif-
concentraby the tissue
B.
55 xi 53 %r 44 v
Perfusion
15 v 15 i? 25 Jii
with
with
A.
PerFusion
OF
CO~CENTRATIDN DIOL/DHT (ng/XJ7K7mL)
COMPARISON
canine
saline
plasma
medium
0.17 0.14
0.29 0.20
1.06
0.75
0.26
1.43
0.71
0.15
0.70
15
16
15
55
41
19
0.26
0.28
0.21
0.76
0.75
0.76
0.41
1.20
0.42
2.91
6.09
3.53
uptake
CY
-VS. CANINE
uptake
( Cdiol)t
IN SAL INE MEDIUM
DHT
0.60
CY
DILUTED
1
DIDL
OF THE PERFUS 'ION CF 3H-DIOL/14C-DHT
TABLE
PLASMA
38
77
36
164
189
102
tCDHT)t
62
UzKercher
Perfusions
et al
of normal prostate
The results
of perfusions
concentrations that
the
the
slices. (a)
of
diol
concentration
influence
both
perfused
of
entry
used
is
in Fig.
illustrated
amount of steroid in the perfusion DHT, the
slices
(mean
Therefore, the diol
diol
2,
was not
6 value
released
for
is
2 A. 2 B.
diol
over
340 nM) is
It was also
into
the
the
range
unsaturable. increase
evident did
from the
(Table
cell
and DHT, 0.04
not
prostatic the
of
steroid
This
finding
was observed
with increasing
observed
to be present
It is
the mechanism regulating
slices
by the tissue
2.
with different
100 ng/mL)
steroid
that
where a linear
retained
(15 to
either
the
50 to
DHT would appear
in the
concentrations 2) that,
after
entry
and 0.17
in a diffusible
contrary into
the
respectively). form,
whereas
not.
[diol]
Figure
into
(from
medium.
of
glands
in Table
steroid
therefore,
steroids
concentrations
to
of
fraction
of
of normal prostate
and DHT are presented
It would appear, entry
glands
in
madium
(Us/o/h)
[DHq
in
medium
(Ho/q/h)
Concentration of diol in tissue (pg/g tissue) as a function of the concentration of diol perfused (pg/g/h). Concentration of DHT in tissue (1;g/g tissue) as a function of the concentration of DHT perfused (vg/g/h).
AXDROGEN
DYNAMCS
IN DOG PROSTATE
63
McKercher
et al
Moreover,
the
64
always
near
in
tissue
the
diol
unity,
of
to predominate of
the diol
diol
DHT were
perfused,
trations.
However,
PDHT_diol,
higher
by increasing
the
increasing
near
to
since
very
intratissular
high
of
diol
can be observed greatly
of that
reduced
in
The interconversion are also
entry, tissular fusion,
slices
hyperplastic
values gland
obtained
are
In
is
by the appear,
contrast,
the
concentrations
of
DHT concen-
greater of
than
DHT are
over
that
the
obtained
observed
for
of both
in
the diol from
by
diol
and
both
one
DHT at steroids
than in the normal prostate.
3.
It
and DHT were
both
dogs.
androgens
from hyperplastic
with normal tissue if
cas-
castrated of
with slices
dogs,
Table
14 weeks with the diol)
However,
with
illustrated
concentrations
obtained
the
from normal
excised
obtained
and uptake. of
obtained
prostates
in vivo
from those
concentrations the
of
The results
metabolism
DHT to
would
intratissular
perfused
and uptake
and intratissular
(dog treated
significantly
experiments
the entry
decreased.
prostates fer
these
of
of DHT perfused.
Dynamics of diol and DHT in prostates trated dogs and dogs with BPH The results
unity.
concentrations
concentration
the concentration
reaction
Pdiol_DHT
the
was metabolized
factor
even when high
higher,
leading
that
slices.
remains
DHT was several-fold
of
the diol
The oxidative
was
DHT ( Pdiol-DHT)
to
to DHT and was influenced
in the tissue
the
diol
The conversion
DHT.
concentrations.
The uptake uptake
to
of al 1 of
that
converted
than that
steroid
therefore,
factor
indicating
was
was lower
perfused
conversion
did
dif-
with regard
considers the
not
the
end of
were
higher
to
intra-
the in
perthe
.83
25/2500
so/50 50/50
Prostate from castrated dogs
.20 .I3
.83
.75
25/25
2500/30
Normal + 3, 17 -dial (14 weeks)
.60 .65 .68
.71 .60 .7E
15/15 15/50 15/1ao
15/15 100/15 500/15
Normal prostate
.75 .79 .80
Cy
Normal prostate
25/25 2500/30 25/2500
(ng/mL)/(ng/mL)
CONCENTRATIONS IN PERFUSION MEDIUM diol/DHT
Normal prostate
TISSUE SAMPLE
.Ol .Ol
.Ol
.Ol
.D5
.04 .02 .02
.02 .03 .04
.02 .06 .02
p
3
OIOL
.21 -20
.90 0.21 0.19
1.45
1.21
1.32
.72
0.70 0.93 0.88
1.43 1.15 1.93
1.06 0.53 O.hl
.87
.7i 1.09 .85
.88 .76 .88
.89 .83 .83
'Stake
I? 12
3,189
3,328
59
'19 77 94
41 134 989
55 1,341 1,391
cCdiol)t
.I9 .I8
.82 .a0 .80
.76 .63 .73
.75 .80 ‘00
.76 .84 .84
cx
1.22 1.00 1.08 0.48 0.91
l03 .02
1.35 0.94 1.00
1.09 1.24 1.01
1.10 1.14 1.08
Pdiol-DHT
.I9 .I2 .I7
.I6 .20 .21
.I2 .08 .ll
.I4 .I0 .I6
P
DHT
FACTORS, UPTAKE, INTRATISSUE CONCENTRATIONS OF TISSUE FROM NORMAL, CASTRATED OR TREATED DDGS
PDHT-diol
ENTRY, RELEASE, CONVERSION DIDL AND DHT BY PROSTATIC
TABLE
0.56 0.51
3.79 2.88 3.34
3.53 3.91 3.51
3.09 3.86 3.95
2.91 1.28 2.88
uptake
44 45
200 7,510 8,443
102 252 403
1,997
189 419
164 3,400 7,581
($HT)~
66
McKercher
The 21 h-l, value
et al
intracellular results
of
6.3
clearance fusions
not h-l)
of
(IC)
clearance shown) within
was always a given
DHT and diol
did
the
greater
tissue
not
were made with different
of
diol
(mean
than
that
to
of
be significant
steroid
concentrations.
on
application
of
DHT (mean
Variations
sample.
appear
value
in
the
when per-
DI!XUSSIDN The present tracer
study
superfusion
trations
of
is
method described
radioactive
steroids
measured at the isotopic from the ments
start
of
by Giorgi
The data
allow
in “batch”
trations
are
cellular would
that,
to
have shown that gen entry DHT are that
are
siologic
of
that little
conditions.
within
cannot
constants
of
higher
than those
consequence,
and the
extent
since
prostate of
were 90 min experi(25).
be estimated
interconversion,
protein
This binding
the hormones available
steroid
concen-
and lead
present Giorgi
to intra-
endogenously and co-workers
the regulation
conversion
concentrations
(18,21,22).
and the
the amounts of
that
in dog plasma
are higher
same at physiological
enzymes
Concen-
and Gurpide
the superfused
found
in the normal canine
ZOO-fold
metabolizing excess
be of
vitro
of hormones.
than those
and uptake
the
rate
in our experiments,
higher
in
shown in similar
and Malathi
parameters
such as the
concentrations appear
as already
of
the
and the superfusate
which was achieved
(18,Zl)
calculation
and the release
The fact
state,
and co-workers
of
by Gurpide and Welch (17).
the superfusion,
the
the
in the tissue
steady
incubations,
the entry
based
of
within
andro-
testosterone
as at would
of
concentrations
indicate the
to
that
cells
to the cells
are
the in
under phy-
Ah-DROGEN
The plasma canine
protein
plasma is
tracers
is
binding
used instead
greatly
This
metrium. albumin tein
It
steroid
is
laries;
however,
those
effect
is
as well
the
has
been
tissues
with
testosterone
and diffuse
into
The entry was similar
for
found
regulating of
of
both
steroids,
the
simple
of
as
tracers
binding
fraction
through
and
Moses
steroids
locally
into
to pro-
of the
the
(24)
such
capilin
that,
as the
from the binding
the steroids
found
by
illustrated
by Malathi
into
Giorgi
and
hunan
protein
of
our values
more unlikely,
with
(25)
were higher
in
than
of the mechanism
the slices
over
co-workers
for
by the
DHT and
slices
and Gurpide
a wide range testosterone,
tissue
linear
diol
in the medium, is compatible
or,
prostatic
and human prostatic
graphically
concentrations
concentrations
normal canine
however,
and DHT with canine
result,
diffusion,
of
the
free
There was no saturation
authors.
intratissular
steroid
of
passage
by
in human endo-
androgen
the
Giorgi
as found
experiments;
the entry
This
by
uptake
DHT and the diol
concentrations,
22).
a high
binding
only
of the
have been obtained
the canine
during
shown
the entry
the cell.
by these
androstenedione
blood
may dissociate
human BPH superfusion those
as to
67
when diluted
of estrogens
the
assumed that
from
it
results
due to
that,
perfusion,
the perfusion
generally
removed
target
prostate,
is
for
Similar
likely
IN DOG PROSTATE
demonstrate
of saline
during
and transcortin
(27).
studies
diminished.
Gurpide and Welch (17)
DYNAMICS
with
(18,21,
increase
of
increasing
with a mechanism of
a specific
carrier
system
of
high capacity. When the perfusions the entry
and uptake
of
were made with prostate both
the
diol
from a castrated
and DHT were greatly
reduced.
dog, It
McKercher
68
et al
has been demonstrated greatly
diminished
increase
in
the
tissue
from
effect
upon the
centration
that
following relative
of
This histological
steroids
into
can
exceeded
that
of
DHT but not
of
the
in
is
because
elements
in
receptors the
equal
are of
has
that
a large con-
perfused
affect
an
prostatic
intratissular
to
might also
for
binding
the
tissue
that
are
(50
the entry
4 to
of
(25)
diol
the
rarely
have also
shown
DHT but not the diol, tissue
gradient
Since the
have
to
medium
the entry
ability
must depend on specific to exist
while
(18,19,21,23)
from tissue
and unsaturable,
has been reported
that
8 times
of
and androstenedione.
similar
indicate
concentration
human and canine
DHT preferentially
that
steroids
and Gurpide
concentration
is
both
of
levels
Malathi
testosterone
to concentrate
uptake
can concentrate
using
two androgens
cellular
rig/g))
DHT at
slices
a positive
for
the
the medium.
and co-workers
tissue
for
while
human prostatic
demonstrated
partly
stromal
The decrease
(45
concentrate
concentration
Giorgi
of
modification
obtained
perfused
that
(28,29),
receptors
the tissue.
The values tissue
androgen
DHT,and consequently
DHT measured
ng/mL).
the
castration
dog.
uptake
and cytosolic
proportion
a castrated
of
nuclear
in the canine
of
the
intraprostate
(30,31). The a value
is
the fraction
unchanged
from the
tissue.
the canine
prostate
(IS),
Gurpide
(25)
measurable present (17).
for
steroid
by Giorgi
and by Grant and Giorgi DHT is
the human prostate,
a diffusible
On the contrary,
superfused
As reported
amounts (mean a value
in both
of
of
0.17),
is
released
and co-workers (23)
released indicating
and a bound form,
and in accordance
that
for
and Malathi
and
from the tissue
in
that
it
must be
which are in equilibrium
with the results
obtained
by
AXDROGEN
Malathi
and
@$0.06),
Gurpide
probably
because
In our superfusions study
of
Malathi
interconversion over
the
vitro
could
the
more efficient
of
an important
In the for
DHT (6.3
of
This
result,
which
(25)
can
explained
other less result plastic
be
binding steroid in
proteins being
a lower
tissue
is
in
of BPH in viva
(14,33).
(mean of
obtained
of diol
is
lower
could
of
this
be
con-
steroid
is
(14). with
lower
normal
than the with
prostatic IC for
that
strong
binding
in
canine
prostate,
the
15.2
and
why the diol
DHT, the diol
concentration
by the
clearance.
is
explain
accordance
available
canine
DHT under normal physiologic
performed
was always
reaction,
important
also
intracellular
of testosterone
h-l)
more
of
show a
in the
DHT concentration
precursor
with -in
also
Thus,
the
preferred
the reverse
the normal plasma concentration another
in
obtained cells
(12,32).
and could
intraprostatic
perfusions
tissue,
results
quantitatively
the induction
the
than that
is
medium
as in the
pathway is
over
intratissular
testosterone, source
as well
epithelial
coenzymes
of the diol
even if
since
ditions, higher
for
tissue,
diol--,DHT
the
69
retention.
laboratory,
pathway
into
human hyperplastic
prostatic
added
the perfusion
than that
prostatic
released
and DHT, the oxidative
why a higher
Moreover,
canine with
IN DOG PROSTATE
not
intracellular
canine
of
is
its
In our
oxidative
explain
diol
of the reaction
absence
the
following is
one. with
net predominance
prostate,
of
of the diol
reductive
in
of
and Gurpide
incubations
even
the
(25),
DYNAMICS
for
metabolism
The IC of h-l)
of
to
the
diol
Malathi DHT to
which
the
IC
(21 h-l). and Gurpide
receptors
would
and release,
the diol
appears
of
tissue,
or
result
in
and thereby
measured in the hyper-
be lower
than that
found
70
in
McKercher
et al
the normal prostate
diol
by
the
steroids
tissue.
were also
by in vivo
and could High
observed
treatment
and DHT in prostates
obtained
from dogs
with
metabolism
canine
androstenedione
androgen
(21).
These differences
to
higher
content
compared
also
induced
diol
(33,34) of
Further
obtained
of
of
the diol
with
the
of
to
while
the
diol
of
and
that
the
fibromuscular studies
the
of
androgen
thelial
cell
types
present
experimental
tissue
approach
is
reduced
by slices
of
by be
at
higher
and the
IC de-
might be related in
results cell
hunan 8PH is
with estrain
both
metsplasia
the relative cells
the
known that
combined
prostatic
receptors
reported could
well
as basal
and stromal
hyperplastic
found
estrogen
to elucidate
and estrogen been
of
as well
glandular
has
It
similar
testosterone,
the
increased
with the diol
presence
androgens
are
elements
dog.
are necessary of
in
with the human tissue
fibro-muscular
that
with
contrary,
is
prostate,
and DHT,do not dif-
perfused
uptake
canine
have obtained
and metabolism
in the dog by treatment
participation
interconversion
slices
entry
of
of the diol
On the
creases
tribution
two
with those
in vivo
and co-workers
prostatic
concentrations,
the
agree
in the hyperplastic
Giorgi
steroid
increase
the
tissue
concentrations
treated
and release
and DHT (18).
human prostate,
prostate
of
hyperplastic
These results
measured
from the normal gland.
the
concentrations
who measured high
parameters
the entry,
results
uptake of the
BPH.
The other
fer
intratissue
with the diol. (14)
i.e.,
from the higher
in the canine
Moore and co-workers
to induce
result
in
the
(35).
extent uptake
tissues.
useful
laboratory for
the
of and
The dis-
in the two prostatic our
an
epi-
(36). study
The of
the
AMJROGEX
metabolic as
under
behavior normal
of the and
androgens
pathologic
DYl’iAMICS
in each
conditions
prostatic and
71
IN DOG PROSTATE
during
cell
type
as well
hormonal
treat-
ment.
ACKNOULUNXNTS The authors are indebted to Claudette Boudreault and Monique Boire for their excellent secretarial assistance. Financial support of the medical Research Council of Canada and the FCAR, A.rebec,is gratefully acknowledged.
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