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Specific Binding of Prolactin by the Prostate Gland of the Rat and Man
0022-5347 /79/1221-0043$02. 00/0 Vol. 122, July
THE JOURNAL OF UROLOGY
Copyright © 1979 by The Williams & Wilkins Co.
Printed in U.S.A.
SPECIFIC BINDING OF PROLACTIN BY THE PROSTATE GLAND OF THE RAT AND MAN E. J. KEENAN, E. D. KEMP, E. E. RAMSEY, L. B. GARRISON, H. D. PEARSE
AND
C. V. HODGES
From the Departments of Surgery and Pharmacology and the Division of Urology, University of Oregon Health Sciences Center, Portland, Oregon
ABSTRACT
Specific binding sites for 125 iodine-prolactin are present in membrane particles obtained from the rat ventral prostate, human benign prostatic hyperplasia and prostatic adenocarcinoma. In the ventral prostate glands of young rats (1 to 4 months old) specific binding of 125iodine-prolactin is higher than in older animals (>8 months old). Subcellular distribution studies revealed that specific 125 iodine-prolactin binding activity is associated primarily with the 15,000 and 100,000 g particulate membrane fractions of the rat ventral prostate and human prostate glands. In rats between 2 and 4 months old significant increases in the prolactin binding activity in the 100,000 g membrane fraction of the ventral prostate are observed to occur without concomitant increases in prolactin binding activity in the 15,000 g fraction. The level of specific 125 iodine-prolactin binding activity present in the human prostate gland is lower than that observed in the rat ventral prostate gland. Localization of specific prolactin binding sites in the rat ventral prostate and the human prostate gland suggests that prolactin could influence the function of these tissues directly. The role of prolactin in the development and function of the prostate gland remains unresolved. Several studies have demonstrated that this adenohypophyseal hormone may indirectly influence the prostate gland by facilitating testicular androgen biosynthesis. 1- 5 However, other studies have indicated that prolactin also augments the actions of androgens in prostatic tissue and, thus, directly alters prostatic activity.6-13 The possibility that prolactin can directly influence the prostate gland has been strengthened recently after the localization of specific prolactin binding sites or receptors in the ventral prostate gland of the rat. 1 4-18 The present studies examined the subcellular distribution of specific prolactin binding activity in the rat ventral prostate gland and the influence of animal age upon this binding distribution. In addition, human prostatic tissue was evaluated for the presence of specific prolactin binding activity to assess whether prolactin could be involved in regulating the function of the human prostate.
was obtained after centrifugation at 1,500 g for 10 minutes. The resulting supernate was centrifuged at 15,000 g for 20 minutes to obtain a crude mitochondrial/Golgi vesicle fraction. A crude plasma membrane/microsomal fraction was prepared by centrifugation of the 15,000 g supernate at 100,000 g for 90 minutes. After centrifugation the membrane particles were resuspended in 0.05 M. tris-hydrochloric acid buffer (pH= 7.6) containing 10 mM. calcium chloride and were frozen at -76C. Prior freezing of the. membrane particles in the presence of calcium chloride caused the membranes to aggregate upon thawing, which facilitated isolation of these particles by low speed centrifugation (1,500 g for 10 minutes). Aliquots (200 µ,l.) of membrane particles containing 100 to 300 µ,g. protein, as determined according to the procedure of Lowry and associates, 20 were added in triplicate to 2 sets of plastic tubes (12 x 75 mm.) and were maintained in an ice bath. To 1 set of tubes 200 µ,l. 0.05 M. tris-hydrochloric acid0.01 M. calcium chloride buffer containing bovine serum albumin (0.5 per cent final concentration) were added and to the second set of tubes 200 µ,l. of the same buffer containing in addition 500 ng. unlabeled prolactin were added. The binding reactions were initiated by the addition of O.1 ml. 1251-prolactin (100,000 counts per minute, 60 to 80 µ,Ci./ µ,g. protein). Incubations were conducted at 25C for 20 hours, since preliminary studies using membrane particles from the lactating rabbit mammary gland and from the rat ventral prostate gland demonstrated that binding equilibrium was established under these conditions. The binding reactions were terminated by the addition of3 ml. ice cold 0.05 M. tris-hydrochloric acid-0.01 M. calcium chloride buffer containing 0.5 per cent bovine serum albumin. 1251-prolactin that was bound to the membrane particles was separated from free 125 I-prolactin by centrifugation (1,500 g for 20 minutes). Unbound 125 I-prolactin in the supernatant was removed by aspiration. The amount of 125 I-prolactin that was bound to membrane particles was quantitated by gamma counting and was expressed as a percentage of the total 125I-prolactin added to the reaction mixture per 0.1 mg. membrane protein. The difference between the amount of 125I-prolactin bound in the absence (total binding) and in the presence of unlabeled prolactin (non-specific binding) was considered specifically bound 1251prolactin. Specific 1251-prolactin binding data were expressed
METHODS
Ventral prostate glands obtained from albino rats after cervical dislocation and human prostatic tissues obtained after transurethral resection or radical prostatectomy were placed on ice, trimmed of extraneous connective tissue and frozen in liquid nitrogen. Specific binding of 1251-prolactin by prostatic tissues was examined using techniques similar to those described by Shiu and Friesen. 19 Tissues were thawed at O to 4C, minced with a scissors, resuspended in ice cold 0.3 M. sucrose (1:10, weight by volume) and homogenized using a Polytron* homogenizer at a setting of 3.5 (2, 10-second bursts with a 30-second cooling interval). Prostatic membrane particles subsequently were obtained by differential centrifugation. A crude particulate fraction Accepted for publication July 21, 1978. Read at annual meeting of American Urological Association, . . Washington, D. C., May 21-25, 1978. Supported in part by Grant RR00334 from the General Chmcal Research Centers Branch of the Division of Research Resources, National Institutes of Health, Grant 38.17 from the Medical Research Foundation of Oregon and the Pleasants Memorial Prostate Cancer Research Center. * Brinkman Instruments, Cantiague Rd., Westbury, New York. 43
KEENAN AND ASSOCIATES
44
as the mean ± standard error of the mean. Approximately 90 per cent of the total 125I-prolactin binding in the rat ventral prostate and approximately 25 per cent of the total binding in the human prostate gland were specifically bound. Initial studies showed that that dissociation constant for specific prolactin binding by the rat ventral prostate was approximately 3.0 x 10-10 M. as determined by Scatchard analysis21 and is, thus, reflective of the high affinity binding characteristic of a prolactin receptor. 19 Ovine prolactin (NIH-P-SlO, 25.6 IU/mg.) was iodinated using lactoperoxidase according to the method of Frantz and Turkington. 22 After iodination 125I-prolactin was separated from free 125I-prolactin by chromatography on a Sephadex G75 column (0.9 x 15 cm.) equilibrated with 0.05 M. trishydrochloric acid-0.1 per cent bovine serum albumin buffer. 125 I-prolactin was purified further by chromatography using a Sephadex G-100 column (0.9 X 60 cm.). Statistical differences among mean values were established by analysis of variance and subsequent application of a multiple range test.
i
Membrane
Fraction
D 1,5ooxg D 15,oooxg 111100,000 xg
<0.1
t---1 - 2-----i
t---2-4-----i
t - - - > 8----t
Age (Months)
Fm. 2. Influence of age upon level and subcellular distribution of specific 1251-prolactin binding activity in rat ventral prostate.
RESULTS
,
Proloctin Binding Activity_
RAT VENTRAL PROSTATE
The subcellular distribution of specific 125I-prolactin binding by membrane particles obtained from the ventral prostate glands and seminal vesicles of rats 4 to 6 months old is shown in figure 1. Specific binding of 125 I-prolactin in the ventral prostate was detectable in the crude particulate fraction (1,500 g), in the 15,000 g fraction and in the crude plasma membrane fraction (100,000 g). In contrast, specific binding of125 I-prolactin was observed only in the 15,000 g fraction of the seminal vesicles and the level of binding activity was significantly lower than that observed in either the 15,000 or 100,000 g fractions of the ventral prostate gland (p <0.05). In rats 4 to 6 months old there was no difference in the level of specific 125Iprolactin binding observed in the 15,000 g and in the crude plasma membrane fractions but, by comparison, the binding activity present in the crude particulate fraction (1,500 g) was significantly lower (p :'.5:0.05). Figure 2 reveals the influences of age upon the level and the subcellular distribution of specific 125I-prolactin binding by membrane particles obtained from the rat ventral prostate gland. In young animals (1 to 2 months old) specific binding of 125 I-prolactin was present in the 15,000 g and the 100,000 g fractions but was not detectable in the crude particulate fraction (1,500 g). At this age specific 125 I-prolactin binding Proloctin Binding Activity_
RAT 3
Membrane Fracttan
D 1.5ooxg [ [J 15 ,000 x g L2l •
100,000xg
activity was significantly higher in the 15,000 g fraction as compared to the 100,000 g or the crude plasma membrane fraction (p <0.05). In rats 2 to 4 months old 125I-prolactin binding activity in the 15,000 g fraction remained unaltered, while the binding of 125 I-prolactin by the crude plasma membrane fraction (100,000 g) increased significantly (p :'.5:0.05). In addition, detectable specific binding of 125I-prolactin was present in the crude particulate fraction (1,500 g), although the level of binding activity was lower than that observed in either of the higherspeed particulate fractions. Membrane particles obtained from the ventral prostate glands of old animals (>8 months) exhibited significantly lower specific binding of 125I-prolactin as compared to either the young (1 to 2 months old) or 2 to 4-month-old animals. Comparable decreases in specific 125I-prolactin binding activity were observed in the 15,000 and 100,000 g fractions. As was observed in the young animals (1 to 2 months old) no specific binding of 125I-prolactin was detectable in the crude particulate fraction (1,500 g). In the human prostate gland significant levels of specific 125 I-prolactin binding activity (>0.1 per cent/0.1 mg. protein) were observed in 7 of 8 hyperplastic tissues (88 per cent) and in 4 of 7 tissues with adenocarcinoma (57 per cent). Figure 3 shows the subcellular distribution and levels of specific 125 Iprolactin binding activity that were observed in those human prostate tissues containing detectable levels of displaceable 125 I-prolactin binding. The specific binding of 125 I-prolactin in the hyperplastic prostate glands and in the prostate glands with adenocarcinoma was distributed equally between the 15,000 and 100,000 g fractions. Specific binding of125I-prolactin was not detectable in the crude particulate fraction (1,500 g) of the human prostate gland. Likewise specific 125 I-prolactin binding was not present in any subcellular fraction of connective tissue. DISCUSSION
Seminal Vesicles Ventral Prostate Fm. 1. Subcellular distribution of specific 1251-prolactin binding activity in ventral prostate gland and seminal vesicles of rat.
The results of the present studies have demonstrated that specific binding sites for prolactin are present in the rat ventral prostate gland and in the human prostate gland. Localization of specific prolactin binding sites in these tissues further suggests that prolactin influences prostatic function directly, as has been indicated by previous studies that examined prostatic responses to exogenous prolactin.s-13 In the rat ventral prostate gland the specific binding of 125 Iprolactin is detectable in the crude particulate fraction (1,500 g) but is associated primarily with the crude mitochondrial/
45
PROLACTIN BINDING IN PROSTATE GLAND
Proloctin Binding Acti11ity_
HUMAN PROSTATE
~ ,;;
""' .<:: "" i:, ---~
"<
.<::
l
D 1,500
1.0 Membrane Fraction
~
xg
15,000 x g
IIJ 100,000xg
C,
·_§ a': ~ ..s,, ?05
--ct ~ C,
•n"' ~
-~ :,::
~
'--<0.1
<0.1
<0.1
(0.1
<0.1
c::;
0..,
<;} FIG. 3. Specific
125
BPI-I
Carcinoma
Connective Tissue
I-prolactin binding activity in human benign prostatic hyperplasia I.BPH) and in prostatic adenocarcinoma
Golgi fraction and in the crude plasma membrane fragments. Age influences not only the level of specific 1251-prolactin binding in the membrane particles of the rat ventral prostate but also the subcellular distribution of binding activity. ·- ~--··--- 125 I-prolactin binding activity is higher in the venprostate glands of young animals (1 to 4 months old) and decreases significantly in. animals 6 months of age or older. Aragona and Friesen reported previously that specific prolacti.n binding activity in the rat ventral prostate decreases with age. 15 At present, the basis for the age-associated decrease in specific prolactin binding by the rat ventral prostate gland is obscure. Between 1 to 2 months and 2 to 4 months of age marked increases in the specific 125 1-prolactin binding activity are observed in the crude plasma membrane fraction (100,000 g), while binding in the mitochondrial/Golgi vesicle fraction remains high but is unaltered. It is possible that the increased specific binding of prolactin that is associated with the crude plasma membrane fraction represents an increase in density of binding sites in this fraction. Further studies involving Scatchard plot analysis will be required to resolve this point but it is interesting that specific prolactin binding activity is high in the plasma membrane fraction at the time when the prostate gland is developing rapidly. In current studies the precise relationship that exists between the subcellular distribution of prolactin binding activity and growth of the ventral prostate gland is being examined. The actual significance of the subcellular distribution of specific prolactin binding sites is uncertain at present. Binding of prolactin by the membrane particles obtained after centrifugation at 100,000 g may represent binding ofprolactin to plasma membrane fragments. 19 Because of its large molecular size (molecular weight = 23,000) prolactin presumably initiates its actions through an interaction with plasma membrane receptor sites. The presence of specific 1251-prolactin binding sites in the 15,000 g fraction of the rat ventral prostate could represent a storage form of the prolactin receptor, which becomes functional only after integration into the plasma membrane. It is conceivable that the specific prolactin binding activity observed in this fraction is associated within Golgi vesicles. Previous studies have localized binding sites for prolactin in Golgi vesicle membranes obtained from the livers of female rats. 23 Binding of 1251-prolactin to membranes in the crude particulate fraction probably represents 125 I-prolactin binding to the plasma membranes of unbroken cells. However, the binding of prolactin to nuclei in this fraction should be considered as a possibility. Thus, it must be emphasized that the significance of the subcellular distribution of prolactin binding sites in the rat ventral prostate gland will remain unclear until studies are conducted using purified membrane fractions that are characterized by subcellular enzyme markers.
The presence of specific prolactin binding activity in the human prostate gland suggests that prolactin could influence the function of this tissue. It is apparent that the level of prolactin binding activity in the abnormal human prostate gland is lower than that observed in the rat ventral prostate gland. This difference could be related to age, the disease process or to a difference in epithelial cell population size relative to stroma. Alternatively, high levels of endogenous prolactin in the plasma of humans responding to operative stress could reduce significantly the number of available prolactin binding sites in the prostate gland. The present role of prolactin in the normal and abnormal function of the prostate gland remains obscure. The presence of specific binding sites for prolactin in the rat ventral prostate gland and the increases in binding activity that occur during the maturation of this tissue indicate that the direct action of prolactin may involve growth of the prostate gland. Localization of specific prolactin binding sites in the human prostate gland suggests that this tissue also may represent a target organ for prolactin. Ovine prolactin (NIH-P-810) was supplied by the National Institute of Arthritis, Metabolic and Digestive Diseases through the Pituitary Hormone Distribution Program. Ms. Pamela Fitzgerald and Ms. Diana Harry contributed in the preparation of the manuscript. REFERENCES 1.
2.
3.
4.
5.
6. 7.
Bartke, A.: Prolactin changes cholesterol stores in the mouse testis. Nature, 224: 700, 1969. Hafiez, A. A., Philpott, J. E. and Bartke, A.: The role of prolactin in the regulation of testicular function: the effect of prolactin and luteinizing hormone on 3-hydroxysteroid dehydrogenase activity in the testes of mice and rats. J. Endocr., 50: 619, 1971. Hafiez, A. A., Lloyd, C. W. and Bartke, A.: The role ofprolactin in the regulation of testis function: the effects of prolactin and luteinizing hormone on the plasma levels of testosterone and androstenedione in hypophysectomized rats. J. Endocr., 52: 327, 1972. Hafiez, A. A., Bartke, A. and Lloyd, C. W.: The role ofprolactin in the regulation of testis function: the synergistic effects of prolactin and luteinizing hormone on the incorporation of l14C-acetate into testosterone and cholesterol by testes from hypophysectomized rats in vitro. J. Endocr., 53: 223, 1972. Musto, N., Hafiez, A. A. and Bartke, A.: Prolactin increases 17 beta-hydroxysteroid dehydrogenase activity in the testis. Endocrinology, 91: 1106, 1972. Grayhack, J. T., Bunce, P. L., Kearns, J. W. and Scott, W.W.: Influence of the pituitary on prostatic response to androgen in the rat. Johns Hopkins Hosp. Bull., 96: 154, 1955. Chase, M. D., Geschwind, I. I. and Bern, H. A.: Synergistic role of prolactin in response of male rat sex accessories to andro-
46
KEENAN AND ASSOCIATES
gen. Proc. Soc. Exp. Biol. Med., 94: 680, 1957. 8. Antliff, H. R., Prasad, M. R. and Meyer, R. K.: Action of prolactin on seminal vesicles of guinea pig. Proc. Soc. Exp. Biol. Med., 103: 77, 1960. 9. Grayhack, J. T.: Pituitary factors influencing growth of the prostate. Natl. Cane. Inst. Monogr., 12: 189, 1963. 10. Reddi, A. H.: Role of prolactin in the growth and secretory activity of the prostate and other accessory glands of mammals. Gen. Comp. Endocr., suppl., 2: 81, 1969. 11. Moger, W. H. and Geschwind, I. I.: The action of prolactin on the sex accessory glands of the male rat. Proc. Soc. Exp. Biol. Med., 141: 1017, 1972. 12. Keenan, E. J. and Thomas, J. A.: Effects of testosterone and prolactin or growth hormone on the accessory sex organs of castrated mice. J. Endocr., 64: 111, 1975. 13. Thomas, J. A. and Keenan, E. J.: Prolactin influences upon androgen action in male accessory sex organs. Adv. Sex Horm. Res., 2: 425, 1976. 14. Frantz, W. L., Maclndoe, J. H. and Turkington, R. W.: Prolactin receptors: characteristics of the particulate fraction binding activity. J. Endocr., 60: 485, 1974. 15. Aragona, C. and Friesen, H. G.: Specific prolactin binding sites in the prostate and testis ofrats. Endocrinology, 97: 677, 1975.
16. Hanlin, M. L. and Yount, A. P.: Prolactin binding in the rat ventral prostate. Endocr. Res. Comm., 2: 489, 1975. 17. Kledzik, G. S., Marshall, S., Campbell, G. A., Gelato, M. and Meites, J.: Effects of castration, testosterone, estradiol, and prolactin on specific prolactin binding activity in ventral prostate of male rats. Endocrinology, 98: 373, 1976. 18. Barkey, R. J., Shani, J., Amit, T. and Barzilai, D.: Specific binding ofprolactin to seminal vesicle, prostate and testicular homogenates of immature, mature and aged rats. J. Endocr., 74: 163, 1977. 19. Shiu, R. P. and Friesen, H. G.: Properties of a prolactin receptor from the rabbit mammary gland. Biochem. J., 140: 301, 1974. 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with Folin phenol reagent. J. Biol. Chem., 193: 265, 1951. 21. Scatchard, G.: The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci., 51: 660, 1949. 22. Frantz, W. L. and Turkington, R. W.: Formation of biologically active 125 I-prolactin by enzymatic radioiodination. Endocrinology, 91: 1545, 1972. 23. Posner, B. I. and Bergeron, J. J. M.: Intracellular polypeptide hormone receptors. Program of the 58th Annual Meeting of the Endocrine Society, p. 165, 1976.
THE JOURNAL OF UROLOGY
Copyright © 1979 by The Williams & Wilkins Co.
Printed in U.S.A.
SPECIFIC BINDING OF PROLACTIN BY THE PROSTATE GLAND OF THE RAT AND MAN E. J. KEENAN, E. D. KEMP, E. E. RAMSEY, L. B. GARRISON, H. D. PEARSE
AND
C. V. HODGES
From the Departments of Surgery and Pharmacology and the Division of Urology, University of Oregon Health Sciences Center, Portland, Oregon
ABSTRACT
Specific binding sites for 125 iodine-prolactin are present in membrane particles obtained from the rat ventral prostate, human benign prostatic hyperplasia and prostatic adenocarcinoma. In the ventral prostate glands of young rats (1 to 4 months old) specific binding of 125iodine-prolactin is higher than in older animals (>8 months old). Subcellular distribution studies revealed that specific 125 iodine-prolactin binding activity is associated primarily with the 15,000 and 100,000 g particulate membrane fractions of the rat ventral prostate and human prostate glands. In rats between 2 and 4 months old significant increases in the prolactin binding activity in the 100,000 g membrane fraction of the ventral prostate are observed to occur without concomitant increases in prolactin binding activity in the 15,000 g fraction. The level of specific 125 iodine-prolactin binding activity present in the human prostate gland is lower than that observed in the rat ventral prostate gland. Localization of specific prolactin binding sites in the rat ventral prostate and the human prostate gland suggests that prolactin could influence the function of these tissues directly. The role of prolactin in the development and function of the prostate gland remains unresolved. Several studies have demonstrated that this adenohypophyseal hormone may indirectly influence the prostate gland by facilitating testicular androgen biosynthesis. 1- 5 However, other studies have indicated that prolactin also augments the actions of androgens in prostatic tissue and, thus, directly alters prostatic activity.6-13 The possibility that prolactin can directly influence the prostate gland has been strengthened recently after the localization of specific prolactin binding sites or receptors in the ventral prostate gland of the rat. 1 4-18 The present studies examined the subcellular distribution of specific prolactin binding activity in the rat ventral prostate gland and the influence of animal age upon this binding distribution. In addition, human prostatic tissue was evaluated for the presence of specific prolactin binding activity to assess whether prolactin could be involved in regulating the function of the human prostate.
was obtained after centrifugation at 1,500 g for 10 minutes. The resulting supernate was centrifuged at 15,000 g for 20 minutes to obtain a crude mitochondrial/Golgi vesicle fraction. A crude plasma membrane/microsomal fraction was prepared by centrifugation of the 15,000 g supernate at 100,000 g for 90 minutes. After centrifugation the membrane particles were resuspended in 0.05 M. tris-hydrochloric acid buffer (pH= 7.6) containing 10 mM. calcium chloride and were frozen at -76C. Prior freezing of the. membrane particles in the presence of calcium chloride caused the membranes to aggregate upon thawing, which facilitated isolation of these particles by low speed centrifugation (1,500 g for 10 minutes). Aliquots (200 µ,l.) of membrane particles containing 100 to 300 µ,g. protein, as determined according to the procedure of Lowry and associates, 20 were added in triplicate to 2 sets of plastic tubes (12 x 75 mm.) and were maintained in an ice bath. To 1 set of tubes 200 µ,l. 0.05 M. tris-hydrochloric acid0.01 M. calcium chloride buffer containing bovine serum albumin (0.5 per cent final concentration) were added and to the second set of tubes 200 µ,l. of the same buffer containing in addition 500 ng. unlabeled prolactin were added. The binding reactions were initiated by the addition of O.1 ml. 1251-prolactin (100,000 counts per minute, 60 to 80 µ,Ci./ µ,g. protein). Incubations were conducted at 25C for 20 hours, since preliminary studies using membrane particles from the lactating rabbit mammary gland and from the rat ventral prostate gland demonstrated that binding equilibrium was established under these conditions. The binding reactions were terminated by the addition of3 ml. ice cold 0.05 M. tris-hydrochloric acid-0.01 M. calcium chloride buffer containing 0.5 per cent bovine serum albumin. 1251-prolactin that was bound to the membrane particles was separated from free 125 I-prolactin by centrifugation (1,500 g for 20 minutes). Unbound 125 I-prolactin in the supernatant was removed by aspiration. The amount of 125 I-prolactin that was bound to membrane particles was quantitated by gamma counting and was expressed as a percentage of the total 125I-prolactin added to the reaction mixture per 0.1 mg. membrane protein. The difference between the amount of 125I-prolactin bound in the absence (total binding) and in the presence of unlabeled prolactin (non-specific binding) was considered specifically bound 1251prolactin. Specific 1251-prolactin binding data were expressed
METHODS
Ventral prostate glands obtained from albino rats after cervical dislocation and human prostatic tissues obtained after transurethral resection or radical prostatectomy were placed on ice, trimmed of extraneous connective tissue and frozen in liquid nitrogen. Specific binding of 1251-prolactin by prostatic tissues was examined using techniques similar to those described by Shiu and Friesen. 19 Tissues were thawed at O to 4C, minced with a scissors, resuspended in ice cold 0.3 M. sucrose (1:10, weight by volume) and homogenized using a Polytron* homogenizer at a setting of 3.5 (2, 10-second bursts with a 30-second cooling interval). Prostatic membrane particles subsequently were obtained by differential centrifugation. A crude particulate fraction Accepted for publication July 21, 1978. Read at annual meeting of American Urological Association, . . Washington, D. C., May 21-25, 1978. Supported in part by Grant RR00334 from the General Chmcal Research Centers Branch of the Division of Research Resources, National Institutes of Health, Grant 38.17 from the Medical Research Foundation of Oregon and the Pleasants Memorial Prostate Cancer Research Center. * Brinkman Instruments, Cantiague Rd., Westbury, New York. 43
KEENAN AND ASSOCIATES
44
as the mean ± standard error of the mean. Approximately 90 per cent of the total 125I-prolactin binding in the rat ventral prostate and approximately 25 per cent of the total binding in the human prostate gland were specifically bound. Initial studies showed that that dissociation constant for specific prolactin binding by the rat ventral prostate was approximately 3.0 x 10-10 M. as determined by Scatchard analysis21 and is, thus, reflective of the high affinity binding characteristic of a prolactin receptor. 19 Ovine prolactin (NIH-P-SlO, 25.6 IU/mg.) was iodinated using lactoperoxidase according to the method of Frantz and Turkington. 22 After iodination 125I-prolactin was separated from free 125I-prolactin by chromatography on a Sephadex G75 column (0.9 x 15 cm.) equilibrated with 0.05 M. trishydrochloric acid-0.1 per cent bovine serum albumin buffer. 125 I-prolactin was purified further by chromatography using a Sephadex G-100 column (0.9 X 60 cm.). Statistical differences among mean values were established by analysis of variance and subsequent application of a multiple range test.
i
Membrane
Fraction
D 1,5ooxg D 15,oooxg 111100,000 xg
<0.1
t---1 - 2-----i
t---2-4-----i
t - - - > 8----t
Age (Months)
Fm. 2. Influence of age upon level and subcellular distribution of specific 1251-prolactin binding activity in rat ventral prostate.
RESULTS
,
Proloctin Binding Activity_
RAT VENTRAL PROSTATE
The subcellular distribution of specific 125I-prolactin binding by membrane particles obtained from the ventral prostate glands and seminal vesicles of rats 4 to 6 months old is shown in figure 1. Specific binding of 125 I-prolactin in the ventral prostate was detectable in the crude particulate fraction (1,500 g), in the 15,000 g fraction and in the crude plasma membrane fraction (100,000 g). In contrast, specific binding of125 I-prolactin was observed only in the 15,000 g fraction of the seminal vesicles and the level of binding activity was significantly lower than that observed in either the 15,000 or 100,000 g fractions of the ventral prostate gland (p <0.05). In rats 4 to 6 months old there was no difference in the level of specific 125Iprolactin binding observed in the 15,000 g and in the crude plasma membrane fractions but, by comparison, the binding activity present in the crude particulate fraction (1,500 g) was significantly lower (p :'.5:0.05). Figure 2 reveals the influences of age upon the level and the subcellular distribution of specific 125I-prolactin binding by membrane particles obtained from the rat ventral prostate gland. In young animals (1 to 2 months old) specific binding of 125 I-prolactin was present in the 15,000 g and the 100,000 g fractions but was not detectable in the crude particulate fraction (1,500 g). At this age specific 125 I-prolactin binding Proloctin Binding Activity_
RAT 3
Membrane Fracttan
D 1.5ooxg [ [J 15 ,000 x g L2l •
100,000xg
activity was significantly higher in the 15,000 g fraction as compared to the 100,000 g or the crude plasma membrane fraction (p <0.05). In rats 2 to 4 months old 125I-prolactin binding activity in the 15,000 g fraction remained unaltered, while the binding of 125 I-prolactin by the crude plasma membrane fraction (100,000 g) increased significantly (p :'.5:0.05). In addition, detectable specific binding of 125I-prolactin was present in the crude particulate fraction (1,500 g), although the level of binding activity was lower than that observed in either of the higherspeed particulate fractions. Membrane particles obtained from the ventral prostate glands of old animals (>8 months) exhibited significantly lower specific binding of 125I-prolactin as compared to either the young (1 to 2 months old) or 2 to 4-month-old animals. Comparable decreases in specific 125I-prolactin binding activity were observed in the 15,000 and 100,000 g fractions. As was observed in the young animals (1 to 2 months old) no specific binding of 125I-prolactin was detectable in the crude particulate fraction (1,500 g). In the human prostate gland significant levels of specific 125 I-prolactin binding activity (>0.1 per cent/0.1 mg. protein) were observed in 7 of 8 hyperplastic tissues (88 per cent) and in 4 of 7 tissues with adenocarcinoma (57 per cent). Figure 3 shows the subcellular distribution and levels of specific 125 Iprolactin binding activity that were observed in those human prostate tissues containing detectable levels of displaceable 125 I-prolactin binding. The specific binding of 125 I-prolactin in the hyperplastic prostate glands and in the prostate glands with adenocarcinoma was distributed equally between the 15,000 and 100,000 g fractions. Specific binding of125I-prolactin was not detectable in the crude particulate fraction (1,500 g) of the human prostate gland. Likewise specific 125 I-prolactin binding was not present in any subcellular fraction of connective tissue. DISCUSSION
Seminal Vesicles Ventral Prostate Fm. 1. Subcellular distribution of specific 1251-prolactin binding activity in ventral prostate gland and seminal vesicles of rat.
The results of the present studies have demonstrated that specific binding sites for prolactin are present in the rat ventral prostate gland and in the human prostate gland. Localization of specific prolactin binding sites in these tissues further suggests that prolactin influences prostatic function directly, as has been indicated by previous studies that examined prostatic responses to exogenous prolactin.s-13 In the rat ventral prostate gland the specific binding of 125 Iprolactin is detectable in the crude particulate fraction (1,500 g) but is associated primarily with the crude mitochondrial/
45
PROLACTIN BINDING IN PROSTATE GLAND
Proloctin Binding Acti11ity_
HUMAN PROSTATE
~ ,;;
""' .<:: "" i:, ---~
"<
.<::
l
D 1,500
1.0 Membrane Fraction
~
xg
15,000 x g
IIJ 100,000xg
C,
·_§ a': ~ ..s,, ?05
--ct ~ C,
•n"' ~
-~ :,::
~
'--<0.1
<0.1
<0.1
(0.1
<0.1
c::;
0..,
<;} FIG. 3. Specific
125
BPI-I
Carcinoma
Connective Tissue
I-prolactin binding activity in human benign prostatic hyperplasia I.BPH) and in prostatic adenocarcinoma
Golgi fraction and in the crude plasma membrane fragments. Age influences not only the level of specific 1251-prolactin binding in the membrane particles of the rat ventral prostate but also the subcellular distribution of binding activity. ·- ~--··--- 125 I-prolactin binding activity is higher in the venprostate glands of young animals (1 to 4 months old) and decreases significantly in. animals 6 months of age or older. Aragona and Friesen reported previously that specific prolacti.n binding activity in the rat ventral prostate decreases with age. 15 At present, the basis for the age-associated decrease in specific prolactin binding by the rat ventral prostate gland is obscure. Between 1 to 2 months and 2 to 4 months of age marked increases in the specific 125 1-prolactin binding activity are observed in the crude plasma membrane fraction (100,000 g), while binding in the mitochondrial/Golgi vesicle fraction remains high but is unaltered. It is possible that the increased specific binding of prolactin that is associated with the crude plasma membrane fraction represents an increase in density of binding sites in this fraction. Further studies involving Scatchard plot analysis will be required to resolve this point but it is interesting that specific prolactin binding activity is high in the plasma membrane fraction at the time when the prostate gland is developing rapidly. In current studies the precise relationship that exists between the subcellular distribution of prolactin binding activity and growth of the ventral prostate gland is being examined. The actual significance of the subcellular distribution of specific prolactin binding sites is uncertain at present. Binding of prolactin by the membrane particles obtained after centrifugation at 100,000 g may represent binding ofprolactin to plasma membrane fragments. 19 Because of its large molecular size (molecular weight = 23,000) prolactin presumably initiates its actions through an interaction with plasma membrane receptor sites. The presence of specific 1251-prolactin binding sites in the 15,000 g fraction of the rat ventral prostate could represent a storage form of the prolactin receptor, which becomes functional only after integration into the plasma membrane. It is conceivable that the specific prolactin binding activity observed in this fraction is associated within Golgi vesicles. Previous studies have localized binding sites for prolactin in Golgi vesicle membranes obtained from the livers of female rats. 23 Binding of 1251-prolactin to membranes in the crude particulate fraction probably represents 125 I-prolactin binding to the plasma membranes of unbroken cells. However, the binding of prolactin to nuclei in this fraction should be considered as a possibility. Thus, it must be emphasized that the significance of the subcellular distribution of prolactin binding sites in the rat ventral prostate gland will remain unclear until studies are conducted using purified membrane fractions that are characterized by subcellular enzyme markers.
The presence of specific prolactin binding activity in the human prostate gland suggests that prolactin could influence the function of this tissue. It is apparent that the level of prolactin binding activity in the abnormal human prostate gland is lower than that observed in the rat ventral prostate gland. This difference could be related to age, the disease process or to a difference in epithelial cell population size relative to stroma. Alternatively, high levels of endogenous prolactin in the plasma of humans responding to operative stress could reduce significantly the number of available prolactin binding sites in the prostate gland. The present role of prolactin in the normal and abnormal function of the prostate gland remains obscure. The presence of specific binding sites for prolactin in the rat ventral prostate gland and the increases in binding activity that occur during the maturation of this tissue indicate that the direct action of prolactin may involve growth of the prostate gland. Localization of specific prolactin binding sites in the human prostate gland suggests that this tissue also may represent a target organ for prolactin. Ovine prolactin (NIH-P-810) was supplied by the National Institute of Arthritis, Metabolic and Digestive Diseases through the Pituitary Hormone Distribution Program. Ms. Pamela Fitzgerald and Ms. Diana Harry contributed in the preparation of the manuscript. REFERENCES 1.
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