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Effect of Estrogens on the Weight and Muscarinic Cholinergic Receptor Density of the Rabbit Bladder and Urethra
0022-534 7/86/1335-1084$02.00/0 Vol. 135, May
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1986 by The Williams & Wilkins Co.
EFFECT OF ESTROGENS ON THE WEIGHT AND MUSCARINIC CHOLINERGIC RECEPTOR DENSITY OF THE RABBIT BLADDER AND URETHRA ELLEN SHAPIRO* From the James Buchanan Brady Urological Institute, Johns Hopkins Hospital and the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland
ABSTRACT
We have determined the muscarinic cholinergic receptor (MChR) density in the adult female rabbit bladder body and bladder base/urethra following three weeks of estrogen treatment. Fifteen female rabbits were separated into three treatment groups. Group I remained intact, Group II underwent bilateral ovariectomy, and Group III underwent simultaneous bilateral ovariectomy followed by subcutaneous placement of a 250 mg. estradiol pellet. The weight, total DNA content, and MChR density were determined for each of the tissues in the three treatment groups. The mean weight of the bladder body following ovariectomy and estrogen treatment (Group III) was threefold greater than in the intact rabbits (Group I) (p <0.001) and two-fold greater than in the ovariectomized rabbits (Group II) (p <0.05). Similar increases were observed in the mean weights of the bladder base/urethral segments in the three treatment groups. The mean total DNA content in Group III was also significantly greater than Group I (p <0.01) and Group II (p <0.05) for both the bladder body and bladder base/urethral segment. The MChR density in the three treatment groups was determined using radioligand receptor binding using [3 H]N-methylscopolamine ([3H]NMS). The MChR density in the bladder body (1.72 fmol./gm. DNA) for Group III was significantly decreased compared to the receptor density in both Group I (3.17 fmol./gm. DNA) and Group II (3.14 fmol./gm. DNA) (p <0.05). The binding of [3H] NMS to the MChR in bladder body was of high affinity (Kd = 0.07-0.15 nM) and was unaltered by hormonal treatment. No difference was found in the MChR density in the bladder base/urethral segment in any of the three treatment groups. In this study, we have shown that estrogens increase the weight and modulate the MChR density of the female rabbit bladder body. The ability to modulate neurotransmitter receptor density by sex steroid hormones may have important clinical implications.
The function of the lower urinary tract is mediated by neurotransmitter substances associated with the autonomic nervous system. 1 The activity of the lower urinary tract can be pharmacologically manipulated by neurotransmitter agonists and antagonists or by agents that regulate the density of neurotransmitter receptors. Neurotransmitters receptor density in various target tissues is modulated by sex steroid hormones. 2- 3 The short term effects of sex steroid hormones on neurotransmitter receptor density in the urinary tract have been studied previously in the rabbit bladder. Levin et al. 3 • 4 demonstrated that the administration of estrogens for four days to immature female rabbits resulted in both increased sensitivity of the bladder to muscarinic cholinergic agonists and increased muscarinic cholinergic receptor (MChR) density. Previous investigations have shown that estrogens alter lower urinary tract function in post-menopausal women. 9- 13 The ability to modulate urinary tract function by sex steroid hormones may have many important clinical implications. The mechanism of these hormone-induced effects warrants further investigation. In this study, we examined the effects of long-term estrogen treatment on the MChR in the adult female rabbit bladder body, bladder base, and urethra using a system of constant drug delivery.
Accepted for publication November 25, 1985. * Requests for reprints: James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD 21205. Supported by NIH grants AM-19300 and MH-25951.
MATERIALS AND METHODS
Fifteen mature female New Zealand white rabbits (Bunnyville Farm) weighing approximately four kg. were separated into three treatment groups. Group I remained intact and received no hormonal manipulation; Group II underwent bilateral ovariectomy and received no hormonal replacement; Group HI underwent simultaneous bilateral ovariectomy followed by subcutaneous placement of a 250 mg. estradiol pellet (Innovative Research of America) which provided three weeks of hormone release (100-250 pg./ml. of serum). Ovariectomy was performed through a lower abdominal midline incision using sterile technique with ketamine (35 mg./kg.) and xylazine (5 mg./kg.) anesthesia. The rabbits were rapidly sacrificed by CO 2 suffocation on post-operative day 21. The bladder body was divided at the level of the ureteral orifices, and the bladder base and urethra were dissected to the urethrovaginal junction. The tissues were dissected free of perivesical fat and serosa, weighed, and stored in a liquid nitrogen freezer. Tissue preparation. The bladder body and bladder base/ urethra from each rabbit was individually immersed into liquid nitrogen and pulverized with a Thermovac tissue pulverizer (Redi-lndustries Corp.). Five hundred milligrams of the pulverized bladder body were used for each saturation experiment. Approximately 150 to 200 mg. of pulverized bladder base/ urethra was used to determine specific binding at a saturating concentration of radioligand. All other body fragments were homogenized in 40 ml. of 50 mM sodium potassium phosphate buffer (pH 7.4) with a Polytron PT-10-35 (60 sec.; setting 6,
1084
ESTROGEt
Brinkrnan o,-·"w,.M.rr 1'he bladder !n·,•r.+!,"r vvas homogenized in 10 mL of buffeL This initial homogenate was filtered through fine Nitex mesh gauze in order to remove connective tissue debris, and then rehomogenized with a Dual! glass-glass homogenizer (Kontes Co.). The homogenate was rehomogenized with the Polytron for 60 seconds and again filtered through fine Nitex mesh gauze. An aliquot (1.5 ml.) was stored in the freezer for DNA determination by the method of Burton 14 using calf thymus DNA as the standard. Saturation analysis. Saturation analyses were performed at seven different concentrations (0.02 nM to 1.5 nM) of [3 H]Nmethylscopolamine ( [3 H] NMS) at constant specific activity (84.8 Ci/mmol.; New England Nuclear Corp.). Total binding was determined in 1.0 ml. containing 800 µl. of tissue homogenate, 100 µI. of 50 mM sodium potassium phosphate buffer (pH 7.4) and 100 µl. of [3 H]NMS at varying concentrations. Nonspecific binding was determined in 1.0 ml. containing 800 µl. of tissue homogenate, 100 of 10 µM atropine (J. T. Baker) and 100 µl. of [3 H]NMS at varying concentrations. Total binding and nonspecific binding determinations for each [3H]NMS concentration were performed in triplicate. Specific binding in the bladder base/urethra was determined using a single saturating concentration of [3 H]NMS (1.5 nM). The assay tubes were gently shaken on a Tectator V shaking platform for one hour at room temperature. Steady state was reached by that time. The binding assays were terminated by filtering on Whatman 2.4 cm. GF /B glass fiber filters (Earl Sandbek). The glass discs were washed four times with four ml. of 4C 50 mM sodium potassium phosphate buffer (pH 7.4) under vacuum suction. The glass filter discs were placed in 10 ml. of aqueous accepting scintillation fluid (Amersham-Searle Corp.) and allowed to equilibrate at room temperature for at least eight hours. The scintillation vials were then counted for 10 minutes on a Beckman LS-230 liquid scintillation counter with an average efficiency of 40 per cent. A linear relationship between maximal specific [3 H]NMS binding and tissue concentration was demonstrated. Scatchard analysis of the data was performed15 using a computer assisted method. Serum estradiol determination. Whole blood was collected from each rabbit at the time of sacrifice and allowed to clot at room temperature, and the serum fractions were separated by of serum from each sample centrifugation. Two hundred was extracted with three ml. of an ethyl acetate:hexane (2:1) solution. The organic phase was taken to dryness, and the residue redissolved in 200 µl. of phosphate buffered saline containing 0.1 per cent gelatin (pH 7.0). Twenty-five microliter aliquots in duplicate were then assayed for 17 /3-estradiol content using a commercially available radioimmunoassay kit (125Iestradiol kit, Cat. #1018, Radioassay Systems Laboratories, Inc.). The sensitivity of the kit was 10 pg./ml. Statistical methods. Student's t test 16 was performed to anathe group differences in MChR density, tissue weights and total DNA content. RESULTS
Serum estradiol levels ranged from 100 to 250 pg./mL in the estrogen treated animals. Both the intact and ovariectomized groups had estradiol levels of less than 10 pg./ml. The bladders in the estrogen treated group (Group III) appeared larger and distended when compared to the bladders in the intact (Group I) and ovariectomized (Group II) groups. The mean weights of the bladder body and the bladder base/urethral segment for the treatment groups are compared in figure lA. The bladder weights in the estrogen treated group (Group III) were three-fold greater than in the intact group (p <0.0001) and two-fold greater than in the ovariectomized group (p <0.001). Similarly, the weights of the bladder base/urethra in Group III were two-fold greater than in both the intact and ovariectomized groups (p <0.01). Figure lB shows the total DNA content of the bladder body
5
Bladder Body
Ill
Bladder Bo§e I Url!lthro
4
Intact
Ovariectomized
Estrogen
Treated
* O>
E 2
-C
@)
C
0
u
z
0
0
~ Intact
Ovarieclom i:1:<1d
Estrogen
Treoled Fm. 1. A, weights (mean ± S.E.M.) of the bladder body and the bladder base/urethral segment for intact, ovariectomized, and estrogentreated groups. * Significant difference from values for the intact (p < 0.001) and ovariectomized groups (p < 0.05). ** Significant difference from values for both intact and ovariectomized groups (p < 0.01). B, total DNA content (mean ± S.E.M.) of the bladder body and bladder base/urethra for intact, ovariectomized and estrogen-treated groups. '' Significant difference from values for both intact and ovariectomized groups (p < 0.01). ** Significant difference from values for both intact and ovariectomized groups (p < 0.05).
and bladder base/urethra in each of the treatment groups. The total bladder body DNA content in Group III was increased two-fold compared to Group I and Group II (p <0.01). The DNA content in the bladder base/urethra in Group HI was also increased 1.5 to two-fold compared with Group I and Group II (p <0.05). The MChR binding capacity (Brnaxl of the bladder and bladder base/urethra was determined by radioligand receptor binding methods using [3H]NMS. Representative Scatchard plots for the binding of [3H]NMS in the rabbit bladder body for each treatment group are shown in figure 2. The Scatchard plots were linear, suggesting a single class of [3H]NMS binding sites. The binding of [3 H]NMS to the rabbit bladder body was saturable and of high affinity. The equilibrium dissociation constants (Ka) were consistent with values determined in other genitourinary tissues 17 and were unaltered by hormone trJatment (Group I, 0.14 ± 0.01 nM; Group II; 0.10 ± 0.02 nM; Group III, 0.10 ± 0.01 nM). The concentration of [3 H]NMS binding sites in the bladder bodies of Group I and Group II was similar (3.17 ± 0.59 and 3.14 ± 0.48 fmol./ µg. DNA) respectively. Estrogen administration (Group III) was associated with a 45 per cent decrease in the MChR density (1. 72 ± 0.18 fmol./ µg. DNA) of the bladder body. The MChR content of the bladder base/urethra was determined in each treatment group by measuring the specific [3H] NMS binding at a saturating concentration of [3 HJNMS. The
1086
SHAPIRO
predicted percentage of receptor sites occupied (B) at various ligand concentrations (L) could be calculated using the equation:18 B
=
B[L] K,i + [L]
providing the Kd and ligand concentration are known. Using this equation, a [3H]NMS concentration of 1.5 nM measures greater than 90 per cent of total receptor binding sites. No statistically significant differences were found in the receptor densities of the bladder base/urethra for any of the treatment groups. The total MChR content of the bladder body and bladder base/urethra was calculated in order to determine the relationship between MChR density and the tissue weight (table 1). Estrogen treatment (Group III) resulted in a decrease in MChR density in the bladder body, however, the total receptor content of these large bladders remained unchanged when compared to the intact and ovariectomized groups. The MChR density in the bladder base/urethra following estrogen treatment remained unchanged and the total receptor content was not statistically different in any of the treatment groups. GI
Intact Kd = 0.12 nM r = -0.99
0.3 o Ovariectomized Kd= 0.08nM r = -0.96 A Estrogen Treated
Kd=O.IOnM r = -0.97
,_ 0.2
(I) (I)
I.L
'
,:, C ::::, 0
CD
0.1
[3
30
20
10
H] NMS Bound ( f mo!)
FIG. 2. Representative Scatchard analyses of [3H]NMS binding to MChR of rabbit bladder body from intact, ovariectomized and estrogentreated groups.
TABLE
DISCUSSION
Bladder and urethral smooth muscle are innervated by the autonomic nervous system. 1 The bladder contains a high density of MChR and therefore MChR analogs influence detrusor smooth muscle function experimentally19-21 and clinically. 22 • 23 An agent that modulates detrusor MChR density can potentially alter the function of the bladder. The effects of estrogens on the MChR binding capacity and binding affinity in the rabbit bladder and urethra were investigated in this study. The duration of hormone administration was similar to the duration of estrogen administration in clinical trials in which estrogens were shown to improve urgency, frequency and incontinence in post-menopausal women. 9 The administration of estrogens resulted in increased bladder and urethral mass. Levin et al. 4 observed that estrogen treated rabbits appeared to have larger bladders than untreated rabbits, and Batra and Iosif 27 reported increases in rabbit urethral and bladder weights following estrogen treatment. The increase in bladder and urethral weight is not secondary to estrogen induced water retention since in our study the total bladder and urethral DNA increased concomitantly. It is conceivable that the increased cellular mass of the bladder and urethra may be secondary to interstitial cells or mucosal hyperplasia. Although other investigators have observed that estrogens increase smooth muscle mass, 27 this phenomenon needs to be demonstrated morphometrically. Estrogen administration was associated with a decrease in the MChR density in the rabbit bladder bodies. Decreased receptor density may result in the reduction of detrusor tone and an increase in bladder capacity. Similar functional changes secondary to a decrease in the receptor density of bladders of women placed on estrogen therapy may in part explain the improvement in their lower urinary tract symptoms following three to four weeks of therapy. 9 Estrogen administration has been shown previously to increase MChR density in the rabbit bladder. 4 In this study, female rabbits received estrogens for only four days. The conflicting results may be explained by the different intervals of estrogen administration. It is our impression that the clinical effects of estrogens should be extrapolated from the group of rabbits receiving chronic estrogen replacement. Other investigators suggest that estrogens improve lower urinary tract symptoms by causing urethral mucosal proliferation 13 or by augmenting the urethral response to alphaadrenergic stimulation. 5 • 11 • 26 Maximum urethral closure pressure and functional urethral profile length have not been consistently altered in women who report resolution of their lower urinary tract symptoms following estrogen treatment. 9 The exact role of the alpha receptor in the female lower urinary tract is not clear. 24- 26 Larsson et al. 26 have demonstrated that estrogens induce a two-fold increase in adrenoceptor content in the female rabbit urethra and increase the sensitivity of isolated urethral tissue preparations to norepinephrine. Alterations in urethral function may be a result of estrogen-induced changes in neurotransmitter receptor density. Estrogen administration appears to affect lower urinary tract function and result in clinical improvement in post-menopausal females. Our study suggests that estrogen-induced down regu-
1. Effect of estrogens on rabbit bladder body and bladder base/urethral muscarinic cholinergic receptors
Treatment Group I Intact II Ovariectomized III Estrogen treated
# of Animals 5 5 5
Bladder Body Total Receptor Receptor Content Density (fmol.) (fmol./µg. DNA) 3.17 ± 0.59 3.14 ± 0.48 1.72 ± 0.18*
3422 ± 697 3399 ± 767 3655 ± 218
Bladder Base/Urethra Receptor Total Density Receptor Content (fmol./µg. DNA) (fmol.) 1.05 ± 0.13 1.85 ± 0.33 1.24 ± 0.41
± 72 463 ± 73 598 ± 99 373
The muscarinic cholinergic receptor density in the bladder body (mean ± S.E.M.) was determined by Scatchard analysis of saturation experiments using [ 3 H]NMS. The muscarinic cholinergic receptor density in the bladder base/urethra was determined at a single saturating concentration (1.5 nM) of [3 H]NMS. * Significant difference from values obtained for both the intact (p < 0.02) and ovariectomized (p < 0.05) groups.
ESTROG£Z'~ EFFECT 0;\} 1}lEIGHT AND RECEPTOR DEl\TSITY
lation of the MChR in the bladder may be another mechanism for improved lower urinary tract symptoms. Acknowledgments. The authors wish to thank Mrs. Janice Berry for editorial assistance and Drs. Herbert Lepor, Patrick C. Walsh, Donald S. Coffey, and Evelyn Barrack for reviewing the manuscript. REFERENCES 1. Andersson, K. E. and Sjogren, C.: Aspects of the physiology and
2. 3. 4. 5.
6. 7.
8. 9. 10. 11. 12.
pharmacology of the bladder and urethra. Prog. Neurobiol., 19: 71, 1982. Hodgson, B. J., Dumas, S., Bolling, D. R. and Heesch, C. M.: Effect of estrogen on sensitivity of rabbit bladder and urethra to phenylephrine. Invest. Urol., 16: 67, 1978. Levin, R. M., Jacobowitz, D. J. and Wein, A. J.: Autonomic innervation of rabbit urinary bladder following estrogen administration. Urology, 17: 449, 1980. Levin, R. M., Shofer, F. S. and Wein, A. J.: Estrogen-induced alterations in the autonomic responses of the rabbit urinary bladder. J. Pharmacol. Exp. Ther., 215: 614, 1980. Downie, J. W., Dean, D. M., Carro-Ciampi, G. and Awad, S. A.: A difference in sensitivity in alpha-adrenergic agonists exhibited by detrusor and bladder neck of rabbit. Can. J. Physiol. Pharmacol., 53: 525, 1975. Roberts, J. M., Insel, P. A. and Goldfien, A.: The regulation of myometrial adenoceptors and adrenergic response by sex steroids. Mo!. Pharmacol., 20: 52, 1981. Bleisch, W., Luine, V. N. and Nottebahm, F.: Modification of synapses in androgen-sensitive muscle. I. Hormonal regulation of acetylcholine receptor number in the songbird syrinx. J. Neurosci., 4: 786, 1984. Gejman, P. V. and Cardinali, D. P.: Hormone effects on muscarinic cholinergic binding in bovine and rat sympathetic superior cervical ganglia. Life Sci., 32: 965, 1983. Walter, S., Wolf, H., Barebo, H. and Jensen, H. K.: Urinary incontinence in post-menopausal women treated with estrogen. Urol. Int., 33: 135, 1978. Salmon, U. J., Walter, R. and Geist, S. H.: The use of estrogens in the treatment of dysuria and incontinence in post-menopausal women. Am. J. Obstet. Gynecol., 42: 845, 1941. Schreiter, F., Fuchs, P. and Stockamp, K.: Estrogenic sensitivity of alpha receptors in the urethra musculature. Urol. Int., 31: 13, 1976. Rud, T.: The effects of estrogens and gestagens on the urethral
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
27.
RABBIT BLADDER
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pressure profile of urinary continent and stress incontinent women. Acta Obstet. Gynecol. Scand., 59: 265, 1980. Caine, M. and Raz, S.. The rnle of female hormone in stress incontinence. Presented at the 16th Congress of Societe Internationale d'Urologie. Amsterdam, 1973. Burton, K.: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric examination of deoxyribonucleic acid. Biochem. J., 62: 315, 1956. Scatchard, G.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci., 51: 660, 1949. Steel, R. G. D. and Torrie, J. H.: Principles and Procedures of Statistics: A Biochemical Approach. New York: McGraw-Hill Book Co., p. 60, 1980. Lepor, H. and Kuhar, M. J.: Characterization ofmuscarinic cholinergic receptor binding in the vas deferens, bladder, prostate, and penis of the rabbit. J. Ural., 132: 392, 1984. Segel, I. H.: Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. New York: John Wiley & Sons, p. 19, 1975. Nergardh, A.: Autonomic receptor functions in the lower urinary tract: a survey of recent experimental results. J. Urol., 113: 180, 1975. Ek, A., Andersson, K. E. and Persson, C. G.: Adenoreceptor and cholinoreceptor mediated responses to the isolated human urethra. Scand. J. Urol. Nephrol., 11: 97, 1977. Levin, R. M. and Wein, A. J.: Direct measurement of the anticholinergic activity of a series of pharmacologic compounds on the canine and rabbit urinary bladder. J. Urol., 128: 396, 1982. Lapides, J.: Urecholine regimen for rehabilitating the atonic bladder. J. Ural., 91: 658, 1964. Lapides, J., Friend, C. R., Ajemian, E. P. and Sanda, L. P.: Comparison of action of oral and parental bethanechol chloride upon the urinary bladder. Invest. Ural., 1: 94, 1963. Gosling, J. A., Dixon, J. S. and Lendon, K. B.: The autonomic innervation of the human male and female bladder neck and proximal urethra. J. Urol., 118: 302, 1977. Larsson, B.: Demonstration of alpha-adenoceptors in the rabbit bladder base and urethra with [3H]dihydroergocryptine ligand binding. Acta Pharmacol. Toxicol., 52: 188, 1983. Larsson, B., Andersson, K. E., Batra, S., Mattiasson, A. and Sjogren, C.: Effects of estradiol on norepinephrine-induced contraction, alpha adenoceptor number and norepinephrine content in the female rabbit urethra. J. Pharmacol. Exp. Therap., 119: 557, 1984. Batra, S. C. and Iosif, C. S.: Female urethra: a target for estrogen action. J. Ural., 129: 418, 1983.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1986 by The Williams & Wilkins Co.
EFFECT OF ESTROGENS ON THE WEIGHT AND MUSCARINIC CHOLINERGIC RECEPTOR DENSITY OF THE RABBIT BLADDER AND URETHRA ELLEN SHAPIRO* From the James Buchanan Brady Urological Institute, Johns Hopkins Hospital and the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland
ABSTRACT
We have determined the muscarinic cholinergic receptor (MChR) density in the adult female rabbit bladder body and bladder base/urethra following three weeks of estrogen treatment. Fifteen female rabbits were separated into three treatment groups. Group I remained intact, Group II underwent bilateral ovariectomy, and Group III underwent simultaneous bilateral ovariectomy followed by subcutaneous placement of a 250 mg. estradiol pellet. The weight, total DNA content, and MChR density were determined for each of the tissues in the three treatment groups. The mean weight of the bladder body following ovariectomy and estrogen treatment (Group III) was threefold greater than in the intact rabbits (Group I) (p <0.001) and two-fold greater than in the ovariectomized rabbits (Group II) (p <0.05). Similar increases were observed in the mean weights of the bladder base/urethral segments in the three treatment groups. The mean total DNA content in Group III was also significantly greater than Group I (p <0.01) and Group II (p <0.05) for both the bladder body and bladder base/urethral segment. The MChR density in the three treatment groups was determined using radioligand receptor binding using [3 H]N-methylscopolamine ([3H]NMS). The MChR density in the bladder body (1.72 fmol./gm. DNA) for Group III was significantly decreased compared to the receptor density in both Group I (3.17 fmol./gm. DNA) and Group II (3.14 fmol./gm. DNA) (p <0.05). The binding of [3H] NMS to the MChR in bladder body was of high affinity (Kd = 0.07-0.15 nM) and was unaltered by hormonal treatment. No difference was found in the MChR density in the bladder base/urethral segment in any of the three treatment groups. In this study, we have shown that estrogens increase the weight and modulate the MChR density of the female rabbit bladder body. The ability to modulate neurotransmitter receptor density by sex steroid hormones may have important clinical implications.
The function of the lower urinary tract is mediated by neurotransmitter substances associated with the autonomic nervous system. 1 The activity of the lower urinary tract can be pharmacologically manipulated by neurotransmitter agonists and antagonists or by agents that regulate the density of neurotransmitter receptors. Neurotransmitters receptor density in various target tissues is modulated by sex steroid hormones. 2- 3 The short term effects of sex steroid hormones on neurotransmitter receptor density in the urinary tract have been studied previously in the rabbit bladder. Levin et al. 3 • 4 demonstrated that the administration of estrogens for four days to immature female rabbits resulted in both increased sensitivity of the bladder to muscarinic cholinergic agonists and increased muscarinic cholinergic receptor (MChR) density. Previous investigations have shown that estrogens alter lower urinary tract function in post-menopausal women. 9- 13 The ability to modulate urinary tract function by sex steroid hormones may have many important clinical implications. The mechanism of these hormone-induced effects warrants further investigation. In this study, we examined the effects of long-term estrogen treatment on the MChR in the adult female rabbit bladder body, bladder base, and urethra using a system of constant drug delivery.
Accepted for publication November 25, 1985. * Requests for reprints: James Buchanan Brady Urological Institute, Johns Hopkins Hospital, Baltimore, MD 21205. Supported by NIH grants AM-19300 and MH-25951.
MATERIALS AND METHODS
Fifteen mature female New Zealand white rabbits (Bunnyville Farm) weighing approximately four kg. were separated into three treatment groups. Group I remained intact and received no hormonal manipulation; Group II underwent bilateral ovariectomy and received no hormonal replacement; Group HI underwent simultaneous bilateral ovariectomy followed by subcutaneous placement of a 250 mg. estradiol pellet (Innovative Research of America) which provided three weeks of hormone release (100-250 pg./ml. of serum). Ovariectomy was performed through a lower abdominal midline incision using sterile technique with ketamine (35 mg./kg.) and xylazine (5 mg./kg.) anesthesia. The rabbits were rapidly sacrificed by CO 2 suffocation on post-operative day 21. The bladder body was divided at the level of the ureteral orifices, and the bladder base and urethra were dissected to the urethrovaginal junction. The tissues were dissected free of perivesical fat and serosa, weighed, and stored in a liquid nitrogen freezer. Tissue preparation. The bladder body and bladder base/ urethra from each rabbit was individually immersed into liquid nitrogen and pulverized with a Thermovac tissue pulverizer (Redi-lndustries Corp.). Five hundred milligrams of the pulverized bladder body were used for each saturation experiment. Approximately 150 to 200 mg. of pulverized bladder base/ urethra was used to determine specific binding at a saturating concentration of radioligand. All other body fragments were homogenized in 40 ml. of 50 mM sodium potassium phosphate buffer (pH 7.4) with a Polytron PT-10-35 (60 sec.; setting 6,
1084
ESTROGEt
Brinkrnan o,-·"w,.M.rr 1'he bladder !n·,•r.+!,"r vvas homogenized in 10 mL of buffeL This initial homogenate was filtered through fine Nitex mesh gauze in order to remove connective tissue debris, and then rehomogenized with a Dual! glass-glass homogenizer (Kontes Co.). The homogenate was rehomogenized with the Polytron for 60 seconds and again filtered through fine Nitex mesh gauze. An aliquot (1.5 ml.) was stored in the freezer for DNA determination by the method of Burton 14 using calf thymus DNA as the standard. Saturation analysis. Saturation analyses were performed at seven different concentrations (0.02 nM to 1.5 nM) of [3 H]Nmethylscopolamine ( [3 H] NMS) at constant specific activity (84.8 Ci/mmol.; New England Nuclear Corp.). Total binding was determined in 1.0 ml. containing 800 µl. of tissue homogenate, 100 µI. of 50 mM sodium potassium phosphate buffer (pH 7.4) and 100 µl. of [3 H]NMS at varying concentrations. Nonspecific binding was determined in 1.0 ml. containing 800 µl. of tissue homogenate, 100 of 10 µM atropine (J. T. Baker) and 100 µl. of [3 H]NMS at varying concentrations. Total binding and nonspecific binding determinations for each [3H]NMS concentration were performed in triplicate. Specific binding in the bladder base/urethra was determined using a single saturating concentration of [3 H]NMS (1.5 nM). The assay tubes were gently shaken on a Tectator V shaking platform for one hour at room temperature. Steady state was reached by that time. The binding assays were terminated by filtering on Whatman 2.4 cm. GF /B glass fiber filters (Earl Sandbek). The glass discs were washed four times with four ml. of 4C 50 mM sodium potassium phosphate buffer (pH 7.4) under vacuum suction. The glass filter discs were placed in 10 ml. of aqueous accepting scintillation fluid (Amersham-Searle Corp.) and allowed to equilibrate at room temperature for at least eight hours. The scintillation vials were then counted for 10 minutes on a Beckman LS-230 liquid scintillation counter with an average efficiency of 40 per cent. A linear relationship between maximal specific [3 H]NMS binding and tissue concentration was demonstrated. Scatchard analysis of the data was performed15 using a computer assisted method. Serum estradiol determination. Whole blood was collected from each rabbit at the time of sacrifice and allowed to clot at room temperature, and the serum fractions were separated by of serum from each sample centrifugation. Two hundred was extracted with three ml. of an ethyl acetate:hexane (2:1) solution. The organic phase was taken to dryness, and the residue redissolved in 200 µl. of phosphate buffered saline containing 0.1 per cent gelatin (pH 7.0). Twenty-five microliter aliquots in duplicate were then assayed for 17 /3-estradiol content using a commercially available radioimmunoassay kit (125Iestradiol kit, Cat. #1018, Radioassay Systems Laboratories, Inc.). The sensitivity of the kit was 10 pg./ml. Statistical methods. Student's t test 16 was performed to anathe group differences in MChR density, tissue weights and total DNA content. RESULTS
Serum estradiol levels ranged from 100 to 250 pg./mL in the estrogen treated animals. Both the intact and ovariectomized groups had estradiol levels of less than 10 pg./ml. The bladders in the estrogen treated group (Group III) appeared larger and distended when compared to the bladders in the intact (Group I) and ovariectomized (Group II) groups. The mean weights of the bladder body and the bladder base/urethral segment for the treatment groups are compared in figure lA. The bladder weights in the estrogen treated group (Group III) were three-fold greater than in the intact group (p <0.0001) and two-fold greater than in the ovariectomized group (p <0.001). Similarly, the weights of the bladder base/urethra in Group III were two-fold greater than in both the intact and ovariectomized groups (p <0.01). Figure lB shows the total DNA content of the bladder body
5
Bladder Body
Ill
Bladder Bo§e I Url!lthro
4
Intact
Ovariectomized
Estrogen
Treated
* O>
E 2
-C
@)
C
0
u
z
0
0
~ Intact
Ovarieclom i:1:<1d
Estrogen
Treoled Fm. 1. A, weights (mean ± S.E.M.) of the bladder body and the bladder base/urethral segment for intact, ovariectomized, and estrogentreated groups. * Significant difference from values for the intact (p < 0.001) and ovariectomized groups (p < 0.05). ** Significant difference from values for both intact and ovariectomized groups (p < 0.01). B, total DNA content (mean ± S.E.M.) of the bladder body and bladder base/urethra for intact, ovariectomized and estrogen-treated groups. '' Significant difference from values for both intact and ovariectomized groups (p < 0.01). ** Significant difference from values for both intact and ovariectomized groups (p < 0.05).
and bladder base/urethra in each of the treatment groups. The total bladder body DNA content in Group III was increased two-fold compared to Group I and Group II (p <0.01). The DNA content in the bladder base/urethra in Group HI was also increased 1.5 to two-fold compared with Group I and Group II (p <0.05). The MChR binding capacity (Brnaxl of the bladder and bladder base/urethra was determined by radioligand receptor binding methods using [3H]NMS. Representative Scatchard plots for the binding of [3H]NMS in the rabbit bladder body for each treatment group are shown in figure 2. The Scatchard plots were linear, suggesting a single class of [3H]NMS binding sites. The binding of [3 H]NMS to the rabbit bladder body was saturable and of high affinity. The equilibrium dissociation constants (Ka) were consistent with values determined in other genitourinary tissues 17 and were unaltered by hormone trJatment (Group I, 0.14 ± 0.01 nM; Group II; 0.10 ± 0.02 nM; Group III, 0.10 ± 0.01 nM). The concentration of [3 H]NMS binding sites in the bladder bodies of Group I and Group II was similar (3.17 ± 0.59 and 3.14 ± 0.48 fmol./ µg. DNA) respectively. Estrogen administration (Group III) was associated with a 45 per cent decrease in the MChR density (1. 72 ± 0.18 fmol./ µg. DNA) of the bladder body. The MChR content of the bladder base/urethra was determined in each treatment group by measuring the specific [3H] NMS binding at a saturating concentration of [3 HJNMS. The
1086
SHAPIRO
predicted percentage of receptor sites occupied (B) at various ligand concentrations (L) could be calculated using the equation:18 B
=
B[L] K,i + [L]
providing the Kd and ligand concentration are known. Using this equation, a [3H]NMS concentration of 1.5 nM measures greater than 90 per cent of total receptor binding sites. No statistically significant differences were found in the receptor densities of the bladder base/urethra for any of the treatment groups. The total MChR content of the bladder body and bladder base/urethra was calculated in order to determine the relationship between MChR density and the tissue weight (table 1). Estrogen treatment (Group III) resulted in a decrease in MChR density in the bladder body, however, the total receptor content of these large bladders remained unchanged when compared to the intact and ovariectomized groups. The MChR density in the bladder base/urethra following estrogen treatment remained unchanged and the total receptor content was not statistically different in any of the treatment groups. GI
Intact Kd = 0.12 nM r = -0.99
0.3 o Ovariectomized Kd= 0.08nM r = -0.96 A Estrogen Treated
Kd=O.IOnM r = -0.97
,_ 0.2
(I) (I)
I.L
'
,:, C ::::, 0
CD
0.1
[3
30
20
10
H] NMS Bound ( f mo!)
FIG. 2. Representative Scatchard analyses of [3H]NMS binding to MChR of rabbit bladder body from intact, ovariectomized and estrogentreated groups.
TABLE
DISCUSSION
Bladder and urethral smooth muscle are innervated by the autonomic nervous system. 1 The bladder contains a high density of MChR and therefore MChR analogs influence detrusor smooth muscle function experimentally19-21 and clinically. 22 • 23 An agent that modulates detrusor MChR density can potentially alter the function of the bladder. The effects of estrogens on the MChR binding capacity and binding affinity in the rabbit bladder and urethra were investigated in this study. The duration of hormone administration was similar to the duration of estrogen administration in clinical trials in which estrogens were shown to improve urgency, frequency and incontinence in post-menopausal women. 9 The administration of estrogens resulted in increased bladder and urethral mass. Levin et al. 4 observed that estrogen treated rabbits appeared to have larger bladders than untreated rabbits, and Batra and Iosif 27 reported increases in rabbit urethral and bladder weights following estrogen treatment. The increase in bladder and urethral weight is not secondary to estrogen induced water retention since in our study the total bladder and urethral DNA increased concomitantly. It is conceivable that the increased cellular mass of the bladder and urethra may be secondary to interstitial cells or mucosal hyperplasia. Although other investigators have observed that estrogens increase smooth muscle mass, 27 this phenomenon needs to be demonstrated morphometrically. Estrogen administration was associated with a decrease in the MChR density in the rabbit bladder bodies. Decreased receptor density may result in the reduction of detrusor tone and an increase in bladder capacity. Similar functional changes secondary to a decrease in the receptor density of bladders of women placed on estrogen therapy may in part explain the improvement in their lower urinary tract symptoms following three to four weeks of therapy. 9 Estrogen administration has been shown previously to increase MChR density in the rabbit bladder. 4 In this study, female rabbits received estrogens for only four days. The conflicting results may be explained by the different intervals of estrogen administration. It is our impression that the clinical effects of estrogens should be extrapolated from the group of rabbits receiving chronic estrogen replacement. Other investigators suggest that estrogens improve lower urinary tract symptoms by causing urethral mucosal proliferation 13 or by augmenting the urethral response to alphaadrenergic stimulation. 5 • 11 • 26 Maximum urethral closure pressure and functional urethral profile length have not been consistently altered in women who report resolution of their lower urinary tract symptoms following estrogen treatment. 9 The exact role of the alpha receptor in the female lower urinary tract is not clear. 24- 26 Larsson et al. 26 have demonstrated that estrogens induce a two-fold increase in adrenoceptor content in the female rabbit urethra and increase the sensitivity of isolated urethral tissue preparations to norepinephrine. Alterations in urethral function may be a result of estrogen-induced changes in neurotransmitter receptor density. Estrogen administration appears to affect lower urinary tract function and result in clinical improvement in post-menopausal females. Our study suggests that estrogen-induced down regu-
1. Effect of estrogens on rabbit bladder body and bladder base/urethral muscarinic cholinergic receptors
Treatment Group I Intact II Ovariectomized III Estrogen treated
# of Animals 5 5 5
Bladder Body Total Receptor Receptor Content Density (fmol.) (fmol./µg. DNA) 3.17 ± 0.59 3.14 ± 0.48 1.72 ± 0.18*
3422 ± 697 3399 ± 767 3655 ± 218
Bladder Base/Urethra Receptor Total Density Receptor Content (fmol./µg. DNA) (fmol.) 1.05 ± 0.13 1.85 ± 0.33 1.24 ± 0.41
± 72 463 ± 73 598 ± 99 373
The muscarinic cholinergic receptor density in the bladder body (mean ± S.E.M.) was determined by Scatchard analysis of saturation experiments using [ 3 H]NMS. The muscarinic cholinergic receptor density in the bladder base/urethra was determined at a single saturating concentration (1.5 nM) of [3 H]NMS. * Significant difference from values obtained for both the intact (p < 0.02) and ovariectomized (p < 0.05) groups.
ESTROG£Z'~ EFFECT 0;\} 1}lEIGHT AND RECEPTOR DEl\TSITY
lation of the MChR in the bladder may be another mechanism for improved lower urinary tract symptoms. Acknowledgments. The authors wish to thank Mrs. Janice Berry for editorial assistance and Drs. Herbert Lepor, Patrick C. Walsh, Donald S. Coffey, and Evelyn Barrack for reviewing the manuscript. REFERENCES 1. Andersson, K. E. and Sjogren, C.: Aspects of the physiology and
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8. 9. 10. 11. 12.
pharmacology of the bladder and urethra. Prog. Neurobiol., 19: 71, 1982. Hodgson, B. J., Dumas, S., Bolling, D. R. and Heesch, C. M.: Effect of estrogen on sensitivity of rabbit bladder and urethra to phenylephrine. Invest. Urol., 16: 67, 1978. Levin, R. M., Jacobowitz, D. J. and Wein, A. J.: Autonomic innervation of rabbit urinary bladder following estrogen administration. Urology, 17: 449, 1980. Levin, R. M., Shofer, F. S. and Wein, A. J.: Estrogen-induced alterations in the autonomic responses of the rabbit urinary bladder. J. Pharmacol. Exp. Ther., 215: 614, 1980. Downie, J. W., Dean, D. M., Carro-Ciampi, G. and Awad, S. A.: A difference in sensitivity in alpha-adrenergic agonists exhibited by detrusor and bladder neck of rabbit. Can. J. Physiol. Pharmacol., 53: 525, 1975. Roberts, J. M., Insel, P. A. and Goldfien, A.: The regulation of myometrial adenoceptors and adrenergic response by sex steroids. Mo!. Pharmacol., 20: 52, 1981. Bleisch, W., Luine, V. N. and Nottebahm, F.: Modification of synapses in androgen-sensitive muscle. I. Hormonal regulation of acetylcholine receptor number in the songbird syrinx. J. Neurosci., 4: 786, 1984. Gejman, P. V. and Cardinali, D. P.: Hormone effects on muscarinic cholinergic binding in bovine and rat sympathetic superior cervical ganglia. Life Sci., 32: 965, 1983. Walter, S., Wolf, H., Barebo, H. and Jensen, H. K.: Urinary incontinence in post-menopausal women treated with estrogen. Urol. Int., 33: 135, 1978. Salmon, U. J., Walter, R. and Geist, S. H.: The use of estrogens in the treatment of dysuria and incontinence in post-menopausal women. Am. J. Obstet. Gynecol., 42: 845, 1941. Schreiter, F., Fuchs, P. and Stockamp, K.: Estrogenic sensitivity of alpha receptors in the urethra musculature. Urol. Int., 31: 13, 1976. Rud, T.: The effects of estrogens and gestagens on the urethral
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RABBIT BLADDER
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pressure profile of urinary continent and stress incontinent women. Acta Obstet. Gynecol. Scand., 59: 265, 1980. Caine, M. and Raz, S.. The rnle of female hormone in stress incontinence. Presented at the 16th Congress of Societe Internationale d'Urologie. Amsterdam, 1973. Burton, K.: A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric examination of deoxyribonucleic acid. Biochem. J., 62: 315, 1956. Scatchard, G.: The attraction of proteins for small molecules and ions. Ann. N.Y. Acad. Sci., 51: 660, 1949. Steel, R. G. D. and Torrie, J. H.: Principles and Procedures of Statistics: A Biochemical Approach. New York: McGraw-Hill Book Co., p. 60, 1980. Lepor, H. and Kuhar, M. J.: Characterization ofmuscarinic cholinergic receptor binding in the vas deferens, bladder, prostate, and penis of the rabbit. J. Ural., 132: 392, 1984. Segel, I. H.: Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. New York: John Wiley & Sons, p. 19, 1975. Nergardh, A.: Autonomic receptor functions in the lower urinary tract: a survey of recent experimental results. J. Urol., 113: 180, 1975. Ek, A., Andersson, K. E. and Persson, C. G.: Adenoreceptor and cholinoreceptor mediated responses to the isolated human urethra. Scand. J. Urol. Nephrol., 11: 97, 1977. Levin, R. M. and Wein, A. J.: Direct measurement of the anticholinergic activity of a series of pharmacologic compounds on the canine and rabbit urinary bladder. J. Urol., 128: 396, 1982. Lapides, J.: Urecholine regimen for rehabilitating the atonic bladder. J. Ural., 91: 658, 1964. Lapides, J., Friend, C. R., Ajemian, E. P. and Sanda, L. P.: Comparison of action of oral and parental bethanechol chloride upon the urinary bladder. Invest. Ural., 1: 94, 1963. Gosling, J. A., Dixon, J. S. and Lendon, K. B.: The autonomic innervation of the human male and female bladder neck and proximal urethra. J. Urol., 118: 302, 1977. Larsson, B.: Demonstration of alpha-adenoceptors in the rabbit bladder base and urethra with [3H]dihydroergocryptine ligand binding. Acta Pharmacol. Toxicol., 52: 188, 1983. Larsson, B., Andersson, K. E., Batra, S., Mattiasson, A. and Sjogren, C.: Effects of estradiol on norepinephrine-induced contraction, alpha adenoceptor number and norepinephrine content in the female rabbit urethra. J. Pharmacol. Exp. Therap., 119: 557, 1984. Batra, S. C. and Iosif, C. S.: Female urethra: a target for estrogen action. J. Ural., 129: 418, 1983.