Life Sciences, Vol. Printed in the U S A
51, pp.
1211-1215
Pergamon
Press
ORCHIECTOMY UPREGULATES RABBIT PROSTATE PERIPHERAL BENZODIAZEPINE RECEPTORS
Satish Batra~'2, and Jan Alenfall2.
Kabi Pharmacia Oncology1, Lund, Sweden and The Department of Obstetrics and Gynecology2, University Hospital, Lund, Sweden. (Received
in final
form August
3, 1992)
Summary The effect of orchiectomy on peripheral benzodiazepine receptors (PBZr) in the rabbit prostate was studied. The mean PBZr density in the mitochondrial fraction isolated from prostates of intact (non castrated) rabbits was 4066 fmol/mg protein which following castration increased to 7236 fmol/mg protein (p< 0.005). The apparent dissociation constant (KD) of prostatic PBZr was higher in castrated rabbits (4.2 nM) than in intact animals (2.7 nM). These data suggest a role of androgen in the regulation of prostatic PBZr.
Specific benzodiazepine binding receptors (BZr) have been identified in several peripheral organs, such as kidney, cortex (but not medulla) of the adrenal gland, heart, ovary, uterus, prostate and testes. Although peripheral benzodiazepine receptors (PBZr) have also been found in the brain, their regional distribution is different from the central BZr in that they are located in the non neuronal tissue of the brain such as glia and choroid plexus. The PBZr are different from the central receptors not only in their ligand specificity but also in their subcellular distribution. Whereas central BZr are primarily located in the plasma membrane, PBZr are found in highest concentration in the mitochondrial outer membranes (1). These PBZr exhibit a strong affinity, in nanomolar range, for Ro5-4864, a benzodiazepine with agonistic properties, and PK 11195, a isoquinoline carboxamide derivative with antagonistic properties, whereas they possess little affinity for the central-type benzodiazepine clonazepam (2). Although the physiological role of the PBZr has not yet been clearly defined they have been strongly implicated in steroidogenesis (3-5), and as well as in the regulation of cell growth and differentiation (6-8). In addition to the fact that steroidogenic organs such as testis, adrenals and placenta have particularly high levels of PBZr, data of some recently published studies suggest that PBZr are regulated by pituitary and gonadal hormones (9). A study by Fares et al (10) Correspondence: S. Batra. Department of Obstetics and Gynecology, University Hospital, Lund, S-221 85 Sweden. Telefax +46 46 15 78 68.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1212
Peripheral Benzodiazepine
Receptors
Vol. 51, No. 15, 1992
showed that gonadotropin and estrogen treatment caused an increase in PBZr density in ovary and uterus in mature rats, with no change in affinity. This treatment, however, did not alter the PBZr density in the kidney (10). Testosterone treatment of intact male rats led to a decrease in PBZr density in testis but an increase in the adrenal gland. There was, however, no change in density of PBZr in the heart or brain or in the central benzodiazepine receptor (11). Gavish et al (12) reported that chronic treatment with estrogen in adult male rats caused a marked reduction of PBZr in the testis and an up-regulation in the kidney, but no change in PBZr in the heart. Available studies on the influence of sex hormones on PBZr are restricted to one species, the rat, and these were done in intact animals only. In order to examine the effect of androgen withdrawal, on PBZr in the prostate, a target for androgens, we have in the present study compared PBZr in the prostate of castrated and intact animals. Materials and methods Chemicals: Tritiated PK 11195 [N-methyl:H] with a specific activity of 86.2 Ci/mmol was purchased from New England Nuclear. Ro 5-4864 was purchased from Fluka and all other chemicals were purchased from Sigma. Animals: White male rabbits weighing between 2.5-3.0 kg were kept in a controlled environment (temperature 21-23oC, light - dark: 12 h) with pellets and tap water ad libitum. The rabbits were divided at random into two groups. One group was orchiectomized three weeks before the animals were sacrificed while the other group of rabbits was left intact. The rabbits were killed by an overdose of pentobarbitone and the prostates were immediately excised and placed in ice-cold sucrose (0.25 M) - HEPES (10 mM) buffer (pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Membrane preparations: After trimming off any excess of fat and connective tissue, the prostates were thoroughly washed in the above buffer, blotted dry and weighed. Each prostate was homogenized on ice in about 5 volumes of the above buffer with a Polytron homogenizer (PGA 10-35) for three periods of 10 s/g tissue with intermittent cooling periods of 30 s. The homogenate was centrifuged at 1000g for 10 min and the pellet was discarded. The supernatant was centrifuged at 8000g for 15 min and the pellet was washed once, then retained as the mitochondrial fraction (m-fraction). The m-fraction was suspended to give a protein concentration of 1-2 mg/ml in icecold sucrose-HEPES buffer, very lightly sonicated for 3 s, VirSonic 300 with microprobe (Virtis Company Inc., Gardiner, NY), and then stored frozen at -70°C until used. The protein concentration in the fractions was determined by the method of Peterson (13). Binding assays: The binding assay was performed in KC1 (100 mM) - HEPES (20 mM) buffer (pH 7.2) containing 0.3 % bovine serum albumin, pH 7.2, in a total volume of 0.5 ml for each assay tube. The protein concentration ranged from 90-150 #g and the concentration of 3H-PK 11195 in various assays varied between 0.1-15 nM. A second set of assay tubes containing 15 /zM Ro54864 was run in order to measure the non-specific binding. The binding reaction was initiated by the addition of protein and incubation was allowed to proceed for 60 min at 4°C. The reaction was terminated by rapid filtration of the incubation mixture through Whatman GF/F glass fiber filter presoaked in 0.5 % polyethylenimine solution. The filter was immediately washed with 3x4 ml ice-cold KCI (100 mM) - HEPES (20 mM) buffer. Filters were placed in vials containing 10 ml Packard Pico-Fluor TM40 scintillation cocktail and counted 12 hours later in a Packard Tri-carb 4640 Liquid Scintillation Analyzer (Packard Instruments Co, Downers Grove, IL).
Vol.
51, No.
15,
Peripheral Benzodiazepine Receptors
1992
1213
Statistical analysis: Students t-test was used for statistical analysis of the observed differences. Results The saturation curves and Scatchard analysis of 3H-PK 11195 specific binding to prostatic mitochondrial fraction from intact and orchiectomized rabbits are presented in fig 1.
A
2500
2000 0
&
~m 1500 E E
O m
iii
~r~"
1000
.
.
=2.5nM
500 000
.
.
.
500 Bound i
i
5
10
PK
11195
.
,
~000
1500
(fmol/mg i
zooo
2500
proteinl !
15
20
(nM)
7500
B
"$
6000
iii "3.
0
E
4500
O
.6nM
E 3000
g
15oo
m
!
.... i
i
3
6
PK
4o° Bound i
9
11195
3o'oo
,~'. . . .
(fmol/mg
oo
,5'oo
proteinl i
i
12
15
(nM)
Fig. 1 Representative saturation and Scatchard curve (inset) of PK 11195 binding to prostatic m-fractions from (A) intact rabbit and 03) orchiectomized rabbit.
1214
Peripheral
Benzodiazepine
Receptors
Vol. 51, No. 15, 1992
Specific binding was saturable with increasing (0.1-15 nM) concentrations while the non-specific binding was linear over this concentration range. The non specific binding was approximately 20 % of the total binding. Scatchard analysis of the data (inset fig 1) shows a single class of binding sites having high affinity to prostatic m-fractions from intact and orchiectomized rabbits yielding B~x and K Dvalues as shown in Table 1. The mean receptor density in prostatic m-fractions from orchiectomized rabbits was significantly higher than that found in m-fractions from intact rabbits (p
51, pp.
1211-1215
Pergamon
Press
ORCHIECTOMY UPREGULATES RABBIT PROSTATE PERIPHERAL BENZODIAZEPINE RECEPTORS
Satish Batra~'2, and Jan Alenfall2.
Kabi Pharmacia Oncology1, Lund, Sweden and The Department of Obstetrics and Gynecology2, University Hospital, Lund, Sweden. (Received
in final
form August
3, 1992)
Summary The effect of orchiectomy on peripheral benzodiazepine receptors (PBZr) in the rabbit prostate was studied. The mean PBZr density in the mitochondrial fraction isolated from prostates of intact (non castrated) rabbits was 4066 fmol/mg protein which following castration increased to 7236 fmol/mg protein (p< 0.005). The apparent dissociation constant (KD) of prostatic PBZr was higher in castrated rabbits (4.2 nM) than in intact animals (2.7 nM). These data suggest a role of androgen in the regulation of prostatic PBZr.
Specific benzodiazepine binding receptors (BZr) have been identified in several peripheral organs, such as kidney, cortex (but not medulla) of the adrenal gland, heart, ovary, uterus, prostate and testes. Although peripheral benzodiazepine receptors (PBZr) have also been found in the brain, their regional distribution is different from the central BZr in that they are located in the non neuronal tissue of the brain such as glia and choroid plexus. The PBZr are different from the central receptors not only in their ligand specificity but also in their subcellular distribution. Whereas central BZr are primarily located in the plasma membrane, PBZr are found in highest concentration in the mitochondrial outer membranes (1). These PBZr exhibit a strong affinity, in nanomolar range, for Ro5-4864, a benzodiazepine with agonistic properties, and PK 11195, a isoquinoline carboxamide derivative with antagonistic properties, whereas they possess little affinity for the central-type benzodiazepine clonazepam (2). Although the physiological role of the PBZr has not yet been clearly defined they have been strongly implicated in steroidogenesis (3-5), and as well as in the regulation of cell growth and differentiation (6-8). In addition to the fact that steroidogenic organs such as testis, adrenals and placenta have particularly high levels of PBZr, data of some recently published studies suggest that PBZr are regulated by pituitary and gonadal hormones (9). A study by Fares et al (10) Correspondence: S. Batra. Department of Obstetics and Gynecology, University Hospital, Lund, S-221 85 Sweden. Telefax +46 46 15 78 68.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
1212
Peripheral Benzodiazepine
Receptors
Vol. 51, No. 15, 1992
showed that gonadotropin and estrogen treatment caused an increase in PBZr density in ovary and uterus in mature rats, with no change in affinity. This treatment, however, did not alter the PBZr density in the kidney (10). Testosterone treatment of intact male rats led to a decrease in PBZr density in testis but an increase in the adrenal gland. There was, however, no change in density of PBZr in the heart or brain or in the central benzodiazepine receptor (11). Gavish et al (12) reported that chronic treatment with estrogen in adult male rats caused a marked reduction of PBZr in the testis and an up-regulation in the kidney, but no change in PBZr in the heart. Available studies on the influence of sex hormones on PBZr are restricted to one species, the rat, and these were done in intact animals only. In order to examine the effect of androgen withdrawal, on PBZr in the prostate, a target for androgens, we have in the present study compared PBZr in the prostate of castrated and intact animals. Materials and methods Chemicals: Tritiated PK 11195 [N-methyl:H] with a specific activity of 86.2 Ci/mmol was purchased from New England Nuclear. Ro 5-4864 was purchased from Fluka and all other chemicals were purchased from Sigma. Animals: White male rabbits weighing between 2.5-3.0 kg were kept in a controlled environment (temperature 21-23oC, light - dark: 12 h) with pellets and tap water ad libitum. The rabbits were divided at random into two groups. One group was orchiectomized three weeks before the animals were sacrificed while the other group of rabbits was left intact. The rabbits were killed by an overdose of pentobarbitone and the prostates were immediately excised and placed in ice-cold sucrose (0.25 M) - HEPES (10 mM) buffer (pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Membrane preparations: After trimming off any excess of fat and connective tissue, the prostates were thoroughly washed in the above buffer, blotted dry and weighed. Each prostate was homogenized on ice in about 5 volumes of the above buffer with a Polytron homogenizer (PGA 10-35) for three periods of 10 s/g tissue with intermittent cooling periods of 30 s. The homogenate was centrifuged at 1000g for 10 min and the pellet was discarded. The supernatant was centrifuged at 8000g for 15 min and the pellet was washed once, then retained as the mitochondrial fraction (m-fraction). The m-fraction was suspended to give a protein concentration of 1-2 mg/ml in icecold sucrose-HEPES buffer, very lightly sonicated for 3 s, VirSonic 300 with microprobe (Virtis Company Inc., Gardiner, NY), and then stored frozen at -70°C until used. The protein concentration in the fractions was determined by the method of Peterson (13). Binding assays: The binding assay was performed in KC1 (100 mM) - HEPES (20 mM) buffer (pH 7.2) containing 0.3 % bovine serum albumin, pH 7.2, in a total volume of 0.5 ml for each assay tube. The protein concentration ranged from 90-150 #g and the concentration of 3H-PK 11195 in various assays varied between 0.1-15 nM. A second set of assay tubes containing 15 /zM Ro54864 was run in order to measure the non-specific binding. The binding reaction was initiated by the addition of protein and incubation was allowed to proceed for 60 min at 4°C. The reaction was terminated by rapid filtration of the incubation mixture through Whatman GF/F glass fiber filter presoaked in 0.5 % polyethylenimine solution. The filter was immediately washed with 3x4 ml ice-cold KCI (100 mM) - HEPES (20 mM) buffer. Filters were placed in vials containing 10 ml Packard Pico-Fluor TM40 scintillation cocktail and counted 12 hours later in a Packard Tri-carb 4640 Liquid Scintillation Analyzer (Packard Instruments Co, Downers Grove, IL).
Vol.
51, No.
15,
Peripheral Benzodiazepine Receptors
1992
1213
Statistical analysis: Students t-test was used for statistical analysis of the observed differences. Results The saturation curves and Scatchard analysis of 3H-PK 11195 specific binding to prostatic mitochondrial fraction from intact and orchiectomized rabbits are presented in fig 1.
A
2500
2000 0
&
~m 1500 E E
O m
iii
~r~"
1000
.
.
=2.5nM
500 000
.
.
.
500 Bound i
i
5
10
PK
11195
.
,
~000
1500
(fmol/mg i
zooo
2500
proteinl !
15
20
(nM)
7500
B
"$
6000
iii "3.
0
E
4500
O
.6nM
E 3000
g
15oo
m
!
.... i
i
3
6
PK
4o° Bound i
9
11195
3o'oo
,~'. . . .
(fmol/mg
oo
,5'oo
proteinl i
i
12
15
(nM)
Fig. 1 Representative saturation and Scatchard curve (inset) of PK 11195 binding to prostatic m-fractions from (A) intact rabbit and 03) orchiectomized rabbit.
1214
Peripheral
Benzodiazepine
Receptors
Vol. 51, No. 15, 1992
Specific binding was saturable with increasing (0.1-15 nM) concentrations while the non-specific binding was linear over this concentration range. The non specific binding was approximately 20 % of the total binding. Scatchard analysis of the data (inset fig 1) shows a single class of binding sites having high affinity to prostatic m-fractions from intact and orchiectomized rabbits yielding B~x and K Dvalues as shown in Table 1. The mean receptor density in prostatic m-fractions from orchiectomized rabbits was significantly higher than that found in m-fractions from intact rabbits (p
