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Decreased Prostatic Secretory Function in Canine Benign Prostatic Hyperplasia is not Due to Decreased Levels of Muscarinic Cholinergic Receptors
0022-534 7/84/1314-0803$02.00/0 Vol. 131, April Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Williams & Wilkins Co.
DECREASED PROSTATIC SECRETORY FUNCTION IN CANINE BENIGN PROSTATIC HYPERPLASIA IS NOT DUE TO DECREASED LEVELS OF MUSCARINIC CHOLINERGIC RECEPTORS HERBERT LEPOR* AND STEPHEN J. BERRY From the James Buchanan Brady Urowgical Institute, Johns Hopkins Hospital, and the Departments of Urowgy and Population Dynamics, Johns Hopkins University Schools of Medicine and Hygiene and Public Health, Baltimore, Maryland
ABSTRACT
The ejaculatory volume and the prostatic secretory capacity (ml. ejaculate per gm. prostate wet weight) were determined for a group of dogs with normal and hyperplastic prostates. The ejaculatory volume and prostatic secretory capacity in dogs with BPH were decreased by 70 per cent and 80 per cent respectively, compared to dogs with normal prostates. Radioligand receptor binding using [ 3H]N-methylscopolamine, a muscarinic cholinergic antagonist, was performed on a similar group of dogs with normal and hyperplastic prostates. The mean equilibrium dissociation constant for the binding of [3H]N-methylscopolamine to homogenates obtained from normal and hyperplastic prostates was 0.21 nM. and 0.19 nM. respectively, demonstrating that the affinity of the receptor binding sites was not altered by the development of BPH. The tissue density of the muscarinic cholinergic receptors (fmol. per mg. prostate wet weight) and the cellular density of these receptors (fmol. per mg. DNA) were not significantly different in normal and hyperplastic prostates. These data indicate that the dramatic reduction in prostatic secretory capacity associated with canine BPH is not related to changes in the muscarinic cholinergic receptor binding capacity. The secretory capacity of the prostate is decreased in dogs with spontaneous benign prostatic hyperplasia (BPH). 1- 3 The mechanism for the decreased secretory activity in the hyperplastic prostate is unknown. Alteration of the muscarinic cholinergic receptor binding sites or down regulation of these receptors may account for the functional changes in prostatic activity. The objective of this study was to determine if changes in the prostatic muscarinic cholinergic receptors are associated with the development of BPH in the dog. The muscarinic cholinergic receptors were compared in normal and hyperplastic prostates since muscarinic cholinergic drugs are known to alter the rate of prostatic secretion in vivo. 4·5 Until now, prostatic muscarinic cholinergic receptors have been characterized only in the rabbit using radioligand receptor binding methods.6 The advantage of radioligand receptor binding is that the concentration of tissue receptors and the binding properties of the receptor sites are measured directly. 7 MATERIALS AND METHODS
Determination of prostatic secretory capacity. Four healthy young male beagles ranging in age between 29 and 42 months (mean = 33 ± 1 month) and 4 healthy old male beagles ranging in age between 86 and 110 months (mean= 96 ± 6 months) were obtained. The birthdate and pedigree of each dog were known and none of the dogs had been used for prior research experiments. Semen was collected from all dogs on 4 different dates with 2 to 7 day intervals between collections. The ejaculates were obtained by manipulating the genitalia of the dogs in the presence of a mongrel bitch in whom estrus had been induced by a subcutaneous silastic implant releasing 1 mg. 17fJestradiol per day. Preparation of tissue for histology and saturation experiments. Four healthy young beagles ranging in age between 30 and 37 months (mean 33 ± 1 month) and 4 healthy old beagles ranging in age between 86 and 110 months (mean 96 ± 6 months) were Accepted for publication November 15, 1983. Supported by HHS grants AM-19300 and MH-25951. *Requests for reprints: Dept. of Urology, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.
anesthetized with pentobarbital (10 mg. per lb.) and the prostate glands were surgically removed and immediately weighed. The prostates were cleaned of adherent areolar tissue and cut into 3 to 5 mm. cross sections. A complete cross section of the gland was placed into 10 per cent buffered formalin for histologic examination and another cross section for receptor binding experiments was placed into a plastic vial and immediately stored in liquid nitrogen. The specimens for histologic evaluation and receptor binding were obtained from similar levels of the prostate for each dog prostate sectioned. The specimens for histologic examination were embedded in paraffin, mounted and stained with hematoxylin and eosin. A study of these sections by light microscopy was performed by a veterinary pathologist without prior information of the ages of the dogs from which the specimens were obtained. The tissue preparation for radioligand receptor binding assay of muscarinic cholinergic receptors has been described previously.6 Five hundred mg. of dog prostate was immersed into liquid N 2 and pulverized. The tissue fragments were homogenized at 4C in 35 ml. of 50 mM. NaKP04 buffer, pH 7.4, with a Brinkman Polytron and then a Duall glass-glass homogenizer. The homogenates were filtered through Nitex fine mesh gauze, rehomogenized with the Brinkman Polytron and refiltered through the Nitex gauze. All procedures were carried out at 4C. Saturation experiments. Saturation analysis was performed at 7 different concentrations (0.02 to 1.50 nM.) of [3 H]NMS ([3H]N-methylscopolamine, specific activity 84.8 Ci/mmol.) on prostate homogenates from 4 young beagles and 4 old beagles. Total binding was determined in 1 ml. containing 800 µl. of tissue homogenate, 100 µl. of 50 mM. NaKP0 4 buffer, pH 7.4 and 100 µI. of [3H)NMS at varying concentrations. Nonspecific binding was determined in 1 ml. containing 800 µl. of tissue homogenate, 100 µI. of 10 µM. atropine and 100 µI. of [3H] NMS at varying concentrations. Total binding and nonspecific binding determinations for each [3 H]NMS concentration were performed in triplicate. Nonspecific binding averaged 10 per cent of total binding. The cpm corresponding to specific binding was less than 10 per cent of the total counts added to each assay for all concentrations of NMS, indicative of zone A conditions.
804
LEPOR AND BERRY TABLE 1.
Histology Normal BPH Level of significance
Age, prostate weight, ejaculatory volume and prostatic secretory capacity of normal and hyperplastic prostates of the beagle No. 4 4
Age (Months) 33 ± 3 96 ± 6 p < 0.001
Ejaculatory Volume
Prostate Weight (Gm.) 12.6 ± 1.3 18.5 ± 2.0 p < 0.05
Relative
(Ml.)
100 147
13.4 ± 3.1 3.9 ± 1.3 p<0.005
Prostatic Secretory Capacity
Relative
(ML/Gm.)
Relative
100 29
1.1 ± 0.27
100 18
0.20 ± 0.05 p < 0.01
Histologic interpretations were performed by a veterinary pathologist without prior information of ages of dogs from which specimens were obtained. Mean and S.E.M. of age, prostate weight, ejaculate volume and prostatic secretory capacity of normal and hyperplastic prostates are presented. p values were determined by unpaired t test.
The incubation and filtration of the assay tubes have been previously described for muscarinic cholinergic saturation experiments in the prostate. 6 The assay tubes were shaken at 6000 rpm for 1 hour at room temperature. The binding assays were terminated by filtering on Whatman 2.4 cm. GF /B glass fiber filters. The glass filter discs were washed 4 times with ice cold 50 mM. NaKP04 buffer, pH 7.4, under vacuum suction. The glass filter discs were placed in 10 ml. of scintillation fluid and counted for 10 minutes on a Beckman LS-230 scintillation counter with an average efficiency of 40 per cent. A linear relationship between specific binding and tissue concentration was demonstrated for [3H]NMS binding to the dog prostate. Saturation curves, Scatchard plots, Kct, Bmax and the linear correlation coefficient of the observed Scatchard plots were generated by a computer program. The DNA concentrations of the prostatic homogenates were determined by the diphenylamine assay8 in order to express the data as fmol. per mg. DNA.
0.30
A) Normal
0.25
Q) Q)
0.20
@
@
$
'-
'+-
'
-0 C
0.15
:::J
0
..0
0.10
@
•
0.05
0.15
DISCUSSION
Brendler et al. 3 demonstrated that beagles with spontaneous BPH have an 80 per cent decreased prostatic secretory capacity
•
B) BPH
RESULTS
The mean age, prostatic weight, ejaculatory volume and prostatic secretory capacity of 4 beagles with normal prostates and 4 beagles with BPH are presented in table 1. The mean age of the dogs with normal prostates and BPH was 2.8 ± 0.25 years and 8.0 ± 0.5 years, respectively. The mean weight of the normal and hyperplastic prostates was 12.6 ± 1.3 gm. and 18.5 ± 2.0 gm. respectively. The mean ejaculatory volume in the young normal dogs was approximately 3.5 times greater than that of the older dogs (p < 0.05). The prostatic secretory capacity (ml. ejaculate per gm. prostate) of the beagles with BPH was 80 per cent less than that of the young dogs (p < 0.01). Prostate specimens obtained from beagles of similar age, prostate weight and prostatic histology were analyzed by ligand saturation experiments using (3 H)N-methylscopolamine. [3 H] NMS is a muscarinic cholinergic antagonist with high specific activity (84.8 Ci/mmol.) and high affinity for muscarinic cholinergic binding sites. Representative Scatchard plots for the binding of [3H)NMS to a normal and hyperplastic dog prostate homogenate are presented in figure 1. The mean equilibrium dissociation constants, Kct, of the Scatchard plots are presented in table 2. The mean Kct's for the young and old dogs were 0.21 nM. and 0.19 nM. respectively. The average linear correlation coefficients of the Scatchard plots for the young and old dogs were essentially 1 which is characteristic of a single binding site (data not shown). The muscarinic cholinergic receptor concentration, Bmax, was determined from the Scatchard plots. The total prostatic receptor content and the receptor concentration were determined (table 2). The tissue concentration of muscarinic cholinergic receptors was expressed as fmol. per mg. prostate wet weight, whereas the concentration of receptors per cell was expressed as fmol. per mg. DNA. There was no statistically significant difference in the tissue concentration or cellular density of muscarinic receptors between normal and hyperplastic prostates.
Kct = 0.21 nM Bmax=5.0fmol/mg wet weight
e
Kd = 0.22 nM
@ Q)
-' Q)
....
Bmax = 3.2 fmol /mg wet weight
Ell
0.10
"
-0 C
:::J
0
..0
0.05 @
10 [ 3 H]
20
30
40
50
NMS bound (fmol /assay)
FIG. 1. Representative Scatchard plots for [3H]NMS binding to homogenate obtained from normal (A) and hyperplastic (B) beagle prostate.
(ml. ejaculate per gm. prostate) when compared to young normal values. In this study, prostatic secretory capacity was also diminished by 80 per cent in dogs with BPH. The muscarinic cholinergic receptors in these normal and hyperplastic prostates were compared in order to determine if the decreased secretory capacity associated with canine BPH was related to changes in the muscarinic cholinergic receptor. The mean equilibrium dissociation constants for [3H]NMS binding to muscarinic cholinergic receptors were not significantly different in the normal and hyperplastic prostates. The affinity of muscarinic cholinergic binding sites are therefore not altered by the development of BPH. The affinity for the muscarinic receptor in the dog is similar to that observed in the rabbit (0.17 nM.) 6 and human BPH specimens (0.10 nM.) (unpublished data). These data together with the results of this study demonstrate homogeneity of muscarinic cholinergic receptors in normal and hyperplastic prostate homogenates from various species. Substantial muscarinic cholinergic receptor concentration in all dog prostates is consistent with the dramatic in vivo augmentation of prostatic secretion following administration of muscarinic cholinergic drugs such as pilocarpine and urecholine. 4·5 There was no statistically significant difference between the relative receptor density and the total receptor content of
DECRii:ASCD PROSTATIC SECRET'O!tY FlJi'llCTI02\J rN CANit~E EP}f TABLE
Histology Normal
BPH Level of
2. Comparison of th,2 niuscarinic cholinergic receptors in norraal and hyperplastic prostates of tlie beagle
No.
Age (Months)
Prostate Weight (gm.)
4 4
33 ± 1 96 ± 6 p < 0.001
12.4 ± 1.4 20.0 ± 2.6 <0.05
Total Receptor Content
Equilibrium Dissociation Constant (nM.)
(Fmol./Mg. Wet Wt.)
(Fmol./Mg. DNA)
(Pmol./Prostate)
0.21 ± 0.04 0.19 ± 0.03
6.24 ± 0.76 5.42 ± 0.83
80 ± 17 107 ± 21
NS
NS
2161 ± 307 2068 ± 284 NS
Receptor Concentration
NS
significance
Equilibrium dissociation constant, Kd, and receptor concentration of normal and hyperplastic prostates were determined from Scatchard plots of [3 H]NMS binding to prostate homogenates obtained from 4 beagles with normal and 4 beagles with hyperplastic prostates. Mean and S.E.M. of equilibrium dissociation constant, receptor concentration and total receptor content of these beagles are presented. The p values were determined by unpaired t test. Mean differences of equilibrium dissociation constant, receptor concentrations and total receptor content for 2 groups were not statistically significant (NS).
normal and hyperplastic beagle prostates. The diminished prostatic secretory capacity associated with canine BPH is therefore not related to down regulation of muscarinic cholinergic receptors. The altered secretory activity may, however, be attributed to the HH~'J"H"'·' of the neurotransmitter-receptor complex to promote secretion or there could be changes in some other component of the receptor complex. The decreased secretory output of the hyperplastic prostate has been attributed to the direct effects of estradiol. 1• 9 The ejaculatory volume in castrated dogs treated with testosterone and estradiol is decreased despite the observation that prostatic size increases and secretory granules remain prominent. 1 Zirkin and Strandberg10 have demonstrated that the density of secretory granules in cpsu,caw.1 cells of normal and hyperplastic dog prostates are similar. The secretory apparatus in the hyperplastic glandular epithelium therefore appears intact since the formation of secretory granules and the content and of the muscarinic cholinergic receptors are equivalent to those in normal epithelium. The release ofprostatic secretory granules or the transpo1t of water in the hyperplastic prostate may in fact be impaired by hormonal factors such as estradiol. The mechanism for the decreased secretory capacity of dogs with BPH cannot be attributed to changes in the muscarinic cholinergic receptor binding. The ejaculatory volume of the dog is nearly entirely derived from the prostate gland since they do not possess seminal vesicles or bulbourethral glands. 11 In man, the major constituent of the seminal volume is derived from the seminal vesicles, 12 and therefore prostatic secretory capacity cannot be determined fron1 ejaculatory volumes. The relationship between the development of BPH, prostatic secretory capacity and muscarinic cholinergic receptor concentration in man cannot be deterto the ····--"···-, ,.,,~,.,.~,_.,,,-, secretion mined model for Can be <:iASC>~j.J'UHXCCO•..t are not altered
n,c,r,.,..
0
wish to thank Dr. Jchn ,=~ sections and Ms. Ruth would also like thank
Drs. Michael J. Kuhar, Patrick C. Walsh, Donald S. Coffey and Evelyn R. Barrack for reviewing the manuscript. REFERENCES l. Huggins, C. and Sommer, J. H.: Quantitative studies of prostatic secretion. III. Simultaneous measurement of size and secretion of the canine prostate and the interaction of androgenic and estrogenic substances thereon. J. Exp. Med., 97: 663, 1953. 2. Wheaton, L. G., DeKlerk, D. P., Strandberg, J. D. and Coffey, D. S.: Relationship of semen volume to size and disease of the prostate in the beagle. Am. J. Vet. Res., 40: 1325, 1979. 3. Brendler, C. B., Berry, S. J., Ewing, L, L., McCullough, A. R., Cochran, R. C., Strandberg, J. R., Zirkin, B. R., Coffey, D. S., Wheaton, L. G., Hiler, M. L., Niswender, G.D., Scott, W.W. and Walsh, P. C.: Spontaneous benigT1 prostatic hyperplasia in the beagle. J. Clin. Invest., 70: 1114, 1983. 4. Smith, E. R.: The stimulation of canine prostate secretion by parasympathomimetic agents. J. Pharma~ol. Exp, Ther., 164: 312, 1968. 5. Bruschini, H., Schmidt, R. A. and Tanagho, E. A.: Neurologic control of prostatic secretion in the dog, Invest. Urol., 15: 288, 1978. 6. Lepor, H. and Kuhar, M. J.: Characterization ofmuscarinic cholinergic receptor binding in the vas deferens, bladder, prostate and penis of the rabbit. Submitted for publication. 7. Yamamura, H. I., Enna, S. J. and Kuhar, M. J.: Neurotransmitter Receptor Binding, Raven Press, New York, 1978. 8. Coffey, D. S., Shimazaki, J. and Williams-Ashman, H. G.: Polymerization of deoxyribonuc!eotides in relation to androgen-induced prostatic growth. Arch. Biochem. Biophys., 124: 184, 1968. 9. Isaacs, J. T., Isaacs, W. B., Wheaton, L. G. and Coffey, D. S.: Differential effects of estrogen treatment on canine seminal plasma components. Invest. Urol., 17: 495, 1980. 10. Zirkin, B. P. and Strandberg, J. D.: Quantitative changes in the morphology of the canine prostate associated with age and spontaneous benign hyperplasia. In press: Anat. Rec. 11. Huggins, C., Masina, M. H., Eichelberger, L. E. and Wharton, J. D.: ua11t1itat,ve studies of prostatic secretion. I. Characteristics normal secretion: the influence bu,m,10""'", and and androgen ,uub1cw.1uuu Med., 70: 543, 1939. 12. of the biochemistry of human semen, Acta (Suppl.), 19: 66, 1949.
THE JOURNAL OF UROLOGY
Copyright© 1984 by The Williams & Wilkins Co.
DECREASED PROSTATIC SECRETORY FUNCTION IN CANINE BENIGN PROSTATIC HYPERPLASIA IS NOT DUE TO DECREASED LEVELS OF MUSCARINIC CHOLINERGIC RECEPTORS HERBERT LEPOR* AND STEPHEN J. BERRY From the James Buchanan Brady Urowgical Institute, Johns Hopkins Hospital, and the Departments of Urowgy and Population Dynamics, Johns Hopkins University Schools of Medicine and Hygiene and Public Health, Baltimore, Maryland
ABSTRACT
The ejaculatory volume and the prostatic secretory capacity (ml. ejaculate per gm. prostate wet weight) were determined for a group of dogs with normal and hyperplastic prostates. The ejaculatory volume and prostatic secretory capacity in dogs with BPH were decreased by 70 per cent and 80 per cent respectively, compared to dogs with normal prostates. Radioligand receptor binding using [ 3H]N-methylscopolamine, a muscarinic cholinergic antagonist, was performed on a similar group of dogs with normal and hyperplastic prostates. The mean equilibrium dissociation constant for the binding of [3H]N-methylscopolamine to homogenates obtained from normal and hyperplastic prostates was 0.21 nM. and 0.19 nM. respectively, demonstrating that the affinity of the receptor binding sites was not altered by the development of BPH. The tissue density of the muscarinic cholinergic receptors (fmol. per mg. prostate wet weight) and the cellular density of these receptors (fmol. per mg. DNA) were not significantly different in normal and hyperplastic prostates. These data indicate that the dramatic reduction in prostatic secretory capacity associated with canine BPH is not related to changes in the muscarinic cholinergic receptor binding capacity. The secretory capacity of the prostate is decreased in dogs with spontaneous benign prostatic hyperplasia (BPH). 1- 3 The mechanism for the decreased secretory activity in the hyperplastic prostate is unknown. Alteration of the muscarinic cholinergic receptor binding sites or down regulation of these receptors may account for the functional changes in prostatic activity. The objective of this study was to determine if changes in the prostatic muscarinic cholinergic receptors are associated with the development of BPH in the dog. The muscarinic cholinergic receptors were compared in normal and hyperplastic prostates since muscarinic cholinergic drugs are known to alter the rate of prostatic secretion in vivo. 4·5 Until now, prostatic muscarinic cholinergic receptors have been characterized only in the rabbit using radioligand receptor binding methods.6 The advantage of radioligand receptor binding is that the concentration of tissue receptors and the binding properties of the receptor sites are measured directly. 7 MATERIALS AND METHODS
Determination of prostatic secretory capacity. Four healthy young male beagles ranging in age between 29 and 42 months (mean = 33 ± 1 month) and 4 healthy old male beagles ranging in age between 86 and 110 months (mean= 96 ± 6 months) were obtained. The birthdate and pedigree of each dog were known and none of the dogs had been used for prior research experiments. Semen was collected from all dogs on 4 different dates with 2 to 7 day intervals between collections. The ejaculates were obtained by manipulating the genitalia of the dogs in the presence of a mongrel bitch in whom estrus had been induced by a subcutaneous silastic implant releasing 1 mg. 17fJestradiol per day. Preparation of tissue for histology and saturation experiments. Four healthy young beagles ranging in age between 30 and 37 months (mean 33 ± 1 month) and 4 healthy old beagles ranging in age between 86 and 110 months (mean 96 ± 6 months) were Accepted for publication November 15, 1983. Supported by HHS grants AM-19300 and MH-25951. *Requests for reprints: Dept. of Urology, Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21205.
anesthetized with pentobarbital (10 mg. per lb.) and the prostate glands were surgically removed and immediately weighed. The prostates were cleaned of adherent areolar tissue and cut into 3 to 5 mm. cross sections. A complete cross section of the gland was placed into 10 per cent buffered formalin for histologic examination and another cross section for receptor binding experiments was placed into a plastic vial and immediately stored in liquid nitrogen. The specimens for histologic evaluation and receptor binding were obtained from similar levels of the prostate for each dog prostate sectioned. The specimens for histologic examination were embedded in paraffin, mounted and stained with hematoxylin and eosin. A study of these sections by light microscopy was performed by a veterinary pathologist without prior information of the ages of the dogs from which the specimens were obtained. The tissue preparation for radioligand receptor binding assay of muscarinic cholinergic receptors has been described previously.6 Five hundred mg. of dog prostate was immersed into liquid N 2 and pulverized. The tissue fragments were homogenized at 4C in 35 ml. of 50 mM. NaKP04 buffer, pH 7.4, with a Brinkman Polytron and then a Duall glass-glass homogenizer. The homogenates were filtered through Nitex fine mesh gauze, rehomogenized with the Brinkman Polytron and refiltered through the Nitex gauze. All procedures were carried out at 4C. Saturation experiments. Saturation analysis was performed at 7 different concentrations (0.02 to 1.50 nM.) of [3 H]NMS ([3H]N-methylscopolamine, specific activity 84.8 Ci/mmol.) on prostate homogenates from 4 young beagles and 4 old beagles. Total binding was determined in 1 ml. containing 800 µl. of tissue homogenate, 100 µl. of 50 mM. NaKP0 4 buffer, pH 7.4 and 100 µI. of [3H)NMS at varying concentrations. Nonspecific binding was determined in 1 ml. containing 800 µl. of tissue homogenate, 100 µI. of 10 µM. atropine and 100 µI. of [3H] NMS at varying concentrations. Total binding and nonspecific binding determinations for each [3 H]NMS concentration were performed in triplicate. Nonspecific binding averaged 10 per cent of total binding. The cpm corresponding to specific binding was less than 10 per cent of the total counts added to each assay for all concentrations of NMS, indicative of zone A conditions.
804
LEPOR AND BERRY TABLE 1.
Histology Normal BPH Level of significance
Age, prostate weight, ejaculatory volume and prostatic secretory capacity of normal and hyperplastic prostates of the beagle No. 4 4
Age (Months) 33 ± 3 96 ± 6 p < 0.001
Ejaculatory Volume
Prostate Weight (Gm.) 12.6 ± 1.3 18.5 ± 2.0 p < 0.05
Relative
(Ml.)
100 147
13.4 ± 3.1 3.9 ± 1.3 p<0.005
Prostatic Secretory Capacity
Relative
(ML/Gm.)
Relative
100 29
1.1 ± 0.27
100 18
0.20 ± 0.05 p < 0.01
Histologic interpretations were performed by a veterinary pathologist without prior information of ages of dogs from which specimens were obtained. Mean and S.E.M. of age, prostate weight, ejaculate volume and prostatic secretory capacity of normal and hyperplastic prostates are presented. p values were determined by unpaired t test.
The incubation and filtration of the assay tubes have been previously described for muscarinic cholinergic saturation experiments in the prostate. 6 The assay tubes were shaken at 6000 rpm for 1 hour at room temperature. The binding assays were terminated by filtering on Whatman 2.4 cm. GF /B glass fiber filters. The glass filter discs were washed 4 times with ice cold 50 mM. NaKP04 buffer, pH 7.4, under vacuum suction. The glass filter discs were placed in 10 ml. of scintillation fluid and counted for 10 minutes on a Beckman LS-230 scintillation counter with an average efficiency of 40 per cent. A linear relationship between specific binding and tissue concentration was demonstrated for [3H]NMS binding to the dog prostate. Saturation curves, Scatchard plots, Kct, Bmax and the linear correlation coefficient of the observed Scatchard plots were generated by a computer program. The DNA concentrations of the prostatic homogenates were determined by the diphenylamine assay8 in order to express the data as fmol. per mg. DNA.
0.30
A) Normal
0.25
Q) Q)
0.20
@
@
$
'-
'+-
'
-0 C
0.15
:::J
0
..0
0.10
@
•
0.05
0.15
DISCUSSION
Brendler et al. 3 demonstrated that beagles with spontaneous BPH have an 80 per cent decreased prostatic secretory capacity
•
B) BPH
RESULTS
The mean age, prostatic weight, ejaculatory volume and prostatic secretory capacity of 4 beagles with normal prostates and 4 beagles with BPH are presented in table 1. The mean age of the dogs with normal prostates and BPH was 2.8 ± 0.25 years and 8.0 ± 0.5 years, respectively. The mean weight of the normal and hyperplastic prostates was 12.6 ± 1.3 gm. and 18.5 ± 2.0 gm. respectively. The mean ejaculatory volume in the young normal dogs was approximately 3.5 times greater than that of the older dogs (p < 0.05). The prostatic secretory capacity (ml. ejaculate per gm. prostate) of the beagles with BPH was 80 per cent less than that of the young dogs (p < 0.01). Prostate specimens obtained from beagles of similar age, prostate weight and prostatic histology were analyzed by ligand saturation experiments using (3 H)N-methylscopolamine. [3 H] NMS is a muscarinic cholinergic antagonist with high specific activity (84.8 Ci/mmol.) and high affinity for muscarinic cholinergic binding sites. Representative Scatchard plots for the binding of [3H)NMS to a normal and hyperplastic dog prostate homogenate are presented in figure 1. The mean equilibrium dissociation constants, Kct, of the Scatchard plots are presented in table 2. The mean Kct's for the young and old dogs were 0.21 nM. and 0.19 nM. respectively. The average linear correlation coefficients of the Scatchard plots for the young and old dogs were essentially 1 which is characteristic of a single binding site (data not shown). The muscarinic cholinergic receptor concentration, Bmax, was determined from the Scatchard plots. The total prostatic receptor content and the receptor concentration were determined (table 2). The tissue concentration of muscarinic cholinergic receptors was expressed as fmol. per mg. prostate wet weight, whereas the concentration of receptors per cell was expressed as fmol. per mg. DNA. There was no statistically significant difference in the tissue concentration or cellular density of muscarinic receptors between normal and hyperplastic prostates.
Kct = 0.21 nM Bmax=5.0fmol/mg wet weight
e
Kd = 0.22 nM
@ Q)
-' Q)
....
Bmax = 3.2 fmol /mg wet weight
Ell
0.10
"
-0 C
:::J
0
..0
0.05 @
10 [ 3 H]
20
30
40
50
NMS bound (fmol /assay)
FIG. 1. Representative Scatchard plots for [3H]NMS binding to homogenate obtained from normal (A) and hyperplastic (B) beagle prostate.
(ml. ejaculate per gm. prostate) when compared to young normal values. In this study, prostatic secretory capacity was also diminished by 80 per cent in dogs with BPH. The muscarinic cholinergic receptors in these normal and hyperplastic prostates were compared in order to determine if the decreased secretory capacity associated with canine BPH was related to changes in the muscarinic cholinergic receptor. The mean equilibrium dissociation constants for [3H]NMS binding to muscarinic cholinergic receptors were not significantly different in the normal and hyperplastic prostates. The affinity of muscarinic cholinergic binding sites are therefore not altered by the development of BPH. The affinity for the muscarinic receptor in the dog is similar to that observed in the rabbit (0.17 nM.) 6 and human BPH specimens (0.10 nM.) (unpublished data). These data together with the results of this study demonstrate homogeneity of muscarinic cholinergic receptors in normal and hyperplastic prostate homogenates from various species. Substantial muscarinic cholinergic receptor concentration in all dog prostates is consistent with the dramatic in vivo augmentation of prostatic secretion following administration of muscarinic cholinergic drugs such as pilocarpine and urecholine. 4·5 There was no statistically significant difference between the relative receptor density and the total receptor content of
DECRii:ASCD PROSTATIC SECRET'O!tY FlJi'llCTI02\J rN CANit~E EP}f TABLE
Histology Normal
BPH Level of
2. Comparison of th,2 niuscarinic cholinergic receptors in norraal and hyperplastic prostates of tlie beagle
No.
Age (Months)
Prostate Weight (gm.)
4 4
33 ± 1 96 ± 6 p < 0.001
12.4 ± 1.4 20.0 ± 2.6 <0.05
Total Receptor Content
Equilibrium Dissociation Constant (nM.)
(Fmol./Mg. Wet Wt.)
(Fmol./Mg. DNA)
(Pmol./Prostate)
0.21 ± 0.04 0.19 ± 0.03
6.24 ± 0.76 5.42 ± 0.83
80 ± 17 107 ± 21
NS
NS
2161 ± 307 2068 ± 284 NS
Receptor Concentration
NS
significance
Equilibrium dissociation constant, Kd, and receptor concentration of normal and hyperplastic prostates were determined from Scatchard plots of [3 H]NMS binding to prostate homogenates obtained from 4 beagles with normal and 4 beagles with hyperplastic prostates. Mean and S.E.M. of equilibrium dissociation constant, receptor concentration and total receptor content of these beagles are presented. The p values were determined by unpaired t test. Mean differences of equilibrium dissociation constant, receptor concentrations and total receptor content for 2 groups were not statistically significant (NS).
normal and hyperplastic beagle prostates. The diminished prostatic secretory capacity associated with canine BPH is therefore not related to down regulation of muscarinic cholinergic receptors. The altered secretory activity may, however, be attributed to the HH~'J"H"'·' of the neurotransmitter-receptor complex to promote secretion or there could be changes in some other component of the receptor complex. The decreased secretory output of the hyperplastic prostate has been attributed to the direct effects of estradiol. 1• 9 The ejaculatory volume in castrated dogs treated with testosterone and estradiol is decreased despite the observation that prostatic size increases and secretory granules remain prominent. 1 Zirkin and Strandberg10 have demonstrated that the density of secretory granules in cpsu,caw.1 cells of normal and hyperplastic dog prostates are similar. The secretory apparatus in the hyperplastic glandular epithelium therefore appears intact since the formation of secretory granules and the content and of the muscarinic cholinergic receptors are equivalent to those in normal epithelium. The release ofprostatic secretory granules or the transpo1t of water in the hyperplastic prostate may in fact be impaired by hormonal factors such as estradiol. The mechanism for the decreased secretory capacity of dogs with BPH cannot be attributed to changes in the muscarinic cholinergic receptor binding. The ejaculatory volume of the dog is nearly entirely derived from the prostate gland since they do not possess seminal vesicles or bulbourethral glands. 11 In man, the major constituent of the seminal volume is derived from the seminal vesicles, 12 and therefore prostatic secretory capacity cannot be determined fron1 ejaculatory volumes. The relationship between the development of BPH, prostatic secretory capacity and muscarinic cholinergic receptor concentration in man cannot be deterto the ····--"···-, ,.,,~,.,.~,_.,,,-, secretion mined model for Can be <:iASC>~j.J'UHXCCO•..t are not altered
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wish to thank Dr. Jchn ,=~ sections and Ms. Ruth would also like thank
Drs. Michael J. Kuhar, Patrick C. Walsh, Donald S. Coffey and Evelyn R. Barrack for reviewing the manuscript. REFERENCES l. Huggins, C. and Sommer, J. H.: Quantitative studies of prostatic secretion. III. Simultaneous measurement of size and secretion of the canine prostate and the interaction of androgenic and estrogenic substances thereon. J. Exp. Med., 97: 663, 1953. 2. Wheaton, L. G., DeKlerk, D. P., Strandberg, J. D. and Coffey, D. S.: Relationship of semen volume to size and disease of the prostate in the beagle. Am. J. Vet. Res., 40: 1325, 1979. 3. Brendler, C. B., Berry, S. J., Ewing, L, L., McCullough, A. R., Cochran, R. C., Strandberg, J. R., Zirkin, B. R., Coffey, D. S., Wheaton, L. G., Hiler, M. L., Niswender, G.D., Scott, W.W. and Walsh, P. C.: Spontaneous benigT1 prostatic hyperplasia in the beagle. J. Clin. Invest., 70: 1114, 1983. 4. Smith, E. R.: The stimulation of canine prostate secretion by parasympathomimetic agents. J. Pharma~ol. Exp, Ther., 164: 312, 1968. 5. Bruschini, H., Schmidt, R. A. and Tanagho, E. A.: Neurologic control of prostatic secretion in the dog, Invest. Urol., 15: 288, 1978. 6. Lepor, H. and Kuhar, M. J.: Characterization ofmuscarinic cholinergic receptor binding in the vas deferens, bladder, prostate and penis of the rabbit. Submitted for publication. 7. Yamamura, H. I., Enna, S. J. and Kuhar, M. J.: Neurotransmitter Receptor Binding, Raven Press, New York, 1978. 8. Coffey, D. S., Shimazaki, J. and Williams-Ashman, H. G.: Polymerization of deoxyribonuc!eotides in relation to androgen-induced prostatic growth. Arch. Biochem. Biophys., 124: 184, 1968. 9. Isaacs, J. T., Isaacs, W. B., Wheaton, L. G. and Coffey, D. S.: Differential effects of estrogen treatment on canine seminal plasma components. Invest. Urol., 17: 495, 1980. 10. Zirkin, B. P. and Strandberg, J. D.: Quantitative changes in the morphology of the canine prostate associated with age and spontaneous benign hyperplasia. In press: Anat. Rec. 11. Huggins, C., Masina, M. H., Eichelberger, L. E. and Wharton, J. D.: ua11t1itat,ve studies of prostatic secretion. I. Characteristics normal secretion: the influence bu,m,10""'", and and androgen ,uub1cw.1uuu Med., 70: 543, 1939. 12. of the biochemistry of human semen, Acta (Suppl.), 19: 66, 1949.