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Alteration of contractile and regulatory proteins in estrogen-induced hypertrophy of female rabbit bladder
BASIC SCIENCE
ALTERATION OF CONTRACTILE AND REGULATORY PROTEINS IN ESTROGEN-INDUCED HYPERTROPHY OF FEMALE RABBIT BLADDER ALPHA DIAN-YU LIN, ROBERT M. LEVIN, BARRY A. KOGAN, CATHERINE WHITBECK, ROBERT E. LEGGETT, CHRISTINE KEARNS, AND ANITA MANNIKAROTTU
ABSTRACT Objectives. Estrogen is essential to mediate physiologic functions in female bladders. Deficiency of estrogen has been speculated to be an etiologic factor for bladder dysfunction in postmenopausal women. Our previous studies have demonstrated that estrogen supplementation in female rabbits induces a “functional hypertrophy” of the urinary bladder smooth muscle. The present study investigated the alterations in the contractile and regulatory proteins in this model. Methods. Twenty New Zealand white female rabbits were separated into five groups of 4 rabbits each. Group 1 served as the control, groups 2 to 6 underwent ovariectomy (Ovx), and group 2 served as the Ovx without estradiol treatment group. Two weeks after Ovx, groups 3 to 5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous implant for 1, 3, and 7 days, respectively. The expression of the contractile and regulatory proteins, such as myosin light chain kinase, rho-kinase, and caldesmon, was analyzed by Western blotting. Results. The expression of myosin light chain kinase was enhanced by estradiol supplementation. The expression of rho-kinase-alpha was increased significantly (20-fold) after Ovx, which was downregulated after estrogen supplementation. No significant change was seen in rho-kinase-beta after Ovx or estradiol supplementation. The expression of caldesmon isoforms was enhanced by 1-day estradiol supplementation but decreased to lower levels than those of the control group by 3 and 7 days of estrogen treatment. Conclusions. The results of the present study have provided more understanding about the role of the contractile and regulatory proteins in detrusor muscle, in both dysfunctional atrophy induced by Ovx and functional hypertrophy induced by estrogen supplementation. UROLOGY 68: 1139–1143, 2006. © 2006 Elsevier Inc.
I
n women, the genital and urinary tract organs are both sensitive to female sex hormones, perhaps because of their common embryologic origin. It has been established that the alterations in circulating estrogen levels that occur during menstrua-
This material was based on work supported in part by the Office of Research and Development (Medical Research Service) of the Department of Veterans Affairs and in part by National Institutes of Health grant RO-1-DK 067114, as well as by the Capital Region Medical Research Foundation. From the Albany College of Pharmacy; Albany Medical College, Albany, New York; Taichung Poh-Ai Hospital, Taichung, Taiwan; and Stratton Veterans Affairs Medical Center, Albany, New York Reprint requests: Anita S. Mannikarottu, Ph.D., Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208. E-mail: [email protected] Submitted: March 15, 2006, accepted (with revisions): August 22, 2006 © 2006 ELSEVIER INC. ALL RIGHTS RESERVED
tion, pregnancy, and menopause can have marked effects on urogenital organ function.1,2 The postmenopausal estrogenic deficit could be related to many urogenital problems. Even though many investigators have considered menopause to be a major risk factor for incontinence, a direct correlation has never been confirmed.3 However, estrogen has been used clinically for the treatment of urinary incontinence in postmenopausal women.4,5 Alterations of contractile and regulatory proteins have been reported in obstructive bladder in animal models mimicking men with benign prostatic hyperplasia-induced bladder outlet obstruction.6 In response to partial bladder outlet obstruction, the rabbit urinary bladder results, initially, in smooth muscle hypertrophy, increased bladder mass, and remodeling of the bladder wall and contractile dysfunctions. Estrogen supplementation to ovariectomized 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.08.1094 1139
rabbits results in significant bladder hypertrophy of a similar magnitude as that observed after partial outlet obstruction in men.7 Using estradiol supplementation, we had reported on an animal model of estrogen-induced functional hypertrophy.8 In contrast to the dysfunctional hypertrophy of the urinary bladder detrusor smooth muscles induced by bladder outlet obstruction, estrogen supplementation induced an enlarged bladder with greater contractility and an increased smooth muscle/collagen ratio. It is our hypothesis that the contractile and regulatory proteins are altered in the functional hypertrophy model by estrogen supplementation and contribute to the augmented contractility of bladder smooth muscle. The present study investigated the estradiol-induced alterations of those proteins. MATERIAL AND METHODS ANIMALS The Institutional Animal Care and Use Committee of the Stratton Veterans Affairs Medical Center approved this project. Twenty female New Zealand white rabbits weighing 3.5 to 4.0 kg were separated into five groups of 4 rabbits each. Group 1 served as the control group and groups 2 to 5 underwent ovariectomy (Ovx). For Ovx, each rabbit was anesthetized with isoflurane (1% to 3%) and both ovaries were excised through bilateral incisions that were closed with 2-0 silk suture. After 14 days, the group 2 rabbits were killed and the bladders excised. The rabbits in groups 3 to 5 were medicated under anesthesia with 17-beta estradiol (Innovative Research of America, Sarasota, Fla) at a dose of 1 mg/kg/day for 1, 3, and 7 days, respectively, by surgical implantation of an estradiol tablet in the subscapular area.
TISSUE PREPARATION The bladder was excised through a midline incision and opened longitudinally from the base to the dome. Three fullthickness longitudinal strips, about 2 ⫻ 10 mm from the ventral surface, were used for the contractile studies, and the balance of the bladder body was frozen in liquid nitrogen and stored at ⫺80°C for molecular analysis.
CONTRACTILITY STUDIES Each strip was mounted in a 15-mL organ bath containing Tyrode’s solution composed of 124.9 mM NaCl, 2.5 mM KCl, 23.8 mM NaHCO3, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 1.8 mM CaCl2, and 5.5 mM dextrose at 37°C for 2 hours. The buffer was equilibrated with a mixture of 95% oxygen and 5% carbon dioxide. Tension was monitored using a Model D Grass Polygraph and digitized using the Grass Polyview system. Preliminary length-tension studies demonstrated that maximal active tension was obtained at 2 g of passive tension for the control and experimental bladders. The strips were equilibrated for 30 minutes at 2-g resting tension. They were then stimulated at 2, 8, and 32 Hz for 20 seconds with pulses 1 ms in duration at 80 V every 5 minutes. Next, the responses to 1 mM adenosine triphosphate (ATP), 20 M carbachol, and 120 mM KCl were determined. Between the additions of the pharmacologic agents, each tissue strip was washed three times at 15-minute intervals with fresh buffer. 1140
PROTEIN PREPARATION, SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS, AND WESTERN BLOTTING Frozen bladder tissues (100 mg) were pulverized while immersed in liquid nitrogen using a mortar and homogenized in buffer containing 20% glycerol, 50 mM tris-HCl (pH 6.8), 0.5% (vol/vol) Tween-20, and protease inhibitors (0.5 mM phenylmethanesulfonyl fluoride, 2 M pepstatin, 2 M antipain, and 0.1 mg/mL trypsin inhibitor).The homogenate was centrifuged at 38,000g for 15 minutes using a centrifuge (Beckman, GS-15R), and the supernatant was collected. After gently mixing the samples in sodium dodecyl sulfate (final concentration 1%), they were boiled for 4 minutes and centrifuged at 10,000 rpm for 15 minutes. The protein concentration was determined using the DC protein assay kit (BioRad Laboratories, Hercules, Calif). Equal amounts (20 g) of total protein from each group were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (except for caldesmon [CaD], which was in 7.5% gel) and transferred to Immobilon-P membranes with Towbin buffer (25 mM tris, 192 mM glycine, and 20% methanol). The membranes were blocked with 5% nonfat milk in 0.05% Tween-20 in phosphate-buffered saline for 1 hour at 37°C and then incubated with primary antibody, myosin light chain kinase (MLCK, 1:2000), rhokinase (ROK)-alpha (clone 21, (1:500), and ROK-beta (clone 46, 1:250, all three from BD Biosciences, San Jose, Calif), CaD (clone C21; 1:1000, Sigma-Aldrich, St. Louis, Mo), in a shaking incubator. The membranes were washed with buffer (20 mM tris, 500 mM NaCl, and 0.05% Tween 20) and incubated with secondary antibody (horseradish peroxidase-conjugated goat antimouse IgG at 1:10,000) for 1 hour at 37°C. Substrates were visualized by echo-chemiluminescence. Band intensities were scanned and analyzed with a Kodak Image Station 440CF and Kodak ID image analysis software (Scientific Image System, Rochester, NY).
STATISTICAL ANALYSIS The data were analyzed using analysis of variance followed by the Neuman-Keuls test for individual differences using Sigma Stat, version 2.03 (SPSS, Chicago, Illinois). P ⬍0.05 was considered statistically significant.
RESULTS The bladders from the Ovx group showed decreased responses to all forms of stimulation (field stimulation, carbachol, and KCl) compared with those from the control group. At 7 days of estrogen treatment, the contractile responses had increased significantly compared with those in the Ovx group and equaled those of the control group (Fig. 1). Figure 2 shows that the expression of MLCK was enhanced by 1, 3, and 7-day estradiol supplementation. Figure 3 demonstrates that the expression of ROK-alpha increased significantly (20-fold) after Ovx and was downregulated after estrogen supplementation. No significant change was seen in ROK-beta after Ovx or estradiol supplementation (Fig. 4). Figure 5 shows that CaD isoforms (heavy chain and light chain) dysregulated at Ovx, enhanced by 1-day estradiol supplementation, and then decreased to lower levels than in the control group with 3 and 7 days of estrogen treatment. UROLOGY 68 (5), 2006
FIGURE 1. Contractile responses to FS, ATP, carbachol, and KCl. Ovx group showed decreased responses to all stimulations compared with control group. In 7-day group, significantly increased responses were noted after 7 days of estradiol treatment compared with Ovx group. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05.
FIGURE 2. (A) Western blot analyses of MLCK expression in each group. Equal amounts of total extractable proteins (20 g) from control, Ovx, and estrogentreated rabbit bladder smooth muscles were separated by electrophoresis, transferred to membrane, and probed with antibody specific to MLCK, as described. Lane 1, control (normal female rabbit); lane 2, ovariectomized for 2 weeks; lanes 3, 4, and 5, estrogen treated for 1, 3, and 7 days, respectively. Ovx group did not show any significant change in MLCK expression. Note, overexpression of MLCK in estrogentreated groups. (B) Average expression of MLCK in different samples. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05. UROLOGY 68 (5), 2006
FIGURE 3. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to ROK-alpha. Note, 20-fold increase in ROK-alpha expression in Ovx rabbits. (B) Average expression of ROKalpha in different samples. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05.
FIGURE 4. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to ROK-beta in each group. No significant change was seen in expression of ROK-beta in different groups. (B) Average expression of ROK-beta in each group. Each bar represents mean ⫾ SEM for n ⫽ 4.
COMMENT Myosin II is the molecular motor for contraction in all muscle types. In general, the velocity of force generation by the muscle is determined by the ATPase activity of myosin II when it interacts with 1141
FIGURE 5. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to CaD in each group. (B) Average expression of CaD isoforms, h-CaD and l-CaD, in each group. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group, P ⬍0.05.
actin.9 Myosin II in smooth muscle cells is activated by actin when the regulatory light chains (MLC20) are phosphorylated by a Ca2⫹-calmodulin-dependent MLCK.10,11 Dephosphorylation of the MLC20 by Ca2⫹-independent phosphatase (MLCP) lowers the actin-activated ATPase activity of myosin.12 A correlation between myosin phosphorylation-dephosphorylation and contractionrelaxation of the smooth muscle has been previously demonstrated.13,14 Myosin phosphorylation and subsequent force can be maintained by inhibiting MLCP.15 Our study demonstrated that Ovx resulted in decreased bladder contractile function and that the contractility returned to normal after estradiol supplementation. However, the present results revealed that MLCK expression was enhanced by 1, 3, and 7-day estradiol supplementation. Estrogen might have enhanced the MLCK activity so as to upregulate MLC phosphorylation, which increased the following contractile response. MLCP is regulated by the small guanosine triphosphatase RhoA and Rho-associated kinase.16 The inhibition of MLCP by RhoA/ROK is associated with the phosphorylation of the MLCP and with smooth muscle contraction in the absence of a rise in intracellular Ca2⫹ concentrations.17 Thus, ROK-mediated regulation of MLC phosphorylation increases the Ca2⫹ sensitivity of smooth muscle contraction.18 An enhanced RhoA/ROK-mediated signal transduction pathway (ROK-alpha) was found in the Ovx and estradiol-augmented groups. The involvement of this pathway suggests a remodeling in the regulation of myosin phos1142
phorylation, which in turn regulates the actin-myosin interaction and contraction in detrusor muscles. Ovx-induced ROK-alpha overexpression might indicate the compensatory effect of the bladder in response to the Ovx and hypoxic damage, despite the failure to increase contractility. Estrogen-induced ROK-alpha overexpression might indicate the promoted myosin phosphorylation, which in turn upregulates the actin-myosin interaction and contraction in detrusor muscles. In contrast to partial bladder outlet obstruction-induced bladder dysfunction, in which the expression of ROK-beta was increased,19 no significant change was found in the ROK-beta expression among the control, Ovx, and estradiol-supplemented groups in the present study. The actin-associated protein CaD is thought to modulate smooth muscle contraction, regulated through thick myosin filaments by way of phosphorylation of the MLC.20 CaD is expressed as two dominant isoforms, l-CaD and h-CaD (low- and high-molecular sizes, respectively). l-CaD is found in nonmuscle cells of various tissue types and h-CaD is present in smooth muscle cells from all sources.21 h-CaD is bound to thin filaments in smooth muscle where it inhibits the actin-myosin interaction and actomyosin ATPase activity. h-CaD inhibits the in vitro motility of actin filaments over myosin heads.22 The role of l-CaD in contractility or cell motility is less clear, although it has been shown to dissociate actin bundles in the presence of Ca2⫹.23 Ovx induced dysregulation in the expression of CaD isoforms, with greater l-CaD and lower expression of h-CaD compared with the control group. The overexpressed l-CaD may be associated with remodeling of the cytoskeletal structure and that the overexpressed l-CaD displaces h-CaD from the thin filaments and that thin filaments containing l-CaD may interfere with the generation and/or maintenance of the additional force required for compensating the Ovx-induced contractile dysfunction. The expressions of both isoforms of CaD were significantly decreased in the estrogen treatment groups by 3 and 7 days, which may have contributed to the increased contractility seen in those groups. The stabilized equilibrium, which was lower than in the control group, might be associated with the augmented contractile function. In contrast to the obstructed bladder model, the expression of l-CaD and h-CaD in the estrogeninduced functional hypertrophy bladder was downregulated, with a certain stable ratio compared with the normal control bladder. CONCLUSIONS The results of this study have demonstrated estrogen-augmented functional hypertrophy and alterations in the contractile and regulatory proteins. UROLOGY 68 (5), 2006
Future investigation of these pathways might provide more understanding of the proteins and the associated modulations. Targeting the associated pathways in the regulation of detrusor muscle contraction would be interesting. REFERENCES 1. Batra SC, and Iosif CS: Female urethra: a target for estrogen action. J Urol 129: 418 – 420, 1983. 2. McGuire EJ: Pathophysiology of incontinence in elderly women, in O’Donnell PD (Ed): Geriatric Medicine. New York, Little, Brown, 1994, pp 221–228. 3. Tinelli A, Tinelli R, Perrone A, et al: Urinary incontinence in postmenopausal period: clinical and pharmacological treatments. Minerva Ginecol 57: 593– 609, 2005. 4. Faber P, and Heidenreich J: Treatment of stress incontinence with estrogen in postmenopausal women. Urol Int 32: 221–227, 1977. 5. Fantl JA, Wyman JF, and Anderson LR: Postmenopausal urinary incontinence: comparison between nonestrogen-supplemented and estrogen-supplemented woman. Obstet Gynecol 71: 823– 828, 1988. 6. Chacko S, Chang S, Hypolite J, et al: Alteration of contractile and regulatory proteins following partial bladder outlet obstruction. Scand J Urol Nephrol Suppl 215: 26 –36, 2004. 7. Aikawa K, Sugino T, Matsumoto S, et al: The effect of ovariectomy and estradiol on rabbit bladder smooth muscle contraction and morphology. J Urol 170(2 Pt 1): 634 – 637, 2003. 8. Lin AD, Levin R, Kogan B, et al: Estrogen induced hypertrophy is accompanied by increased force generation. Neurourol Urodyn May 10, 2006; Epub ahead of print. 9. Barany M: ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50(suppl 218): 197–218, 1967. 10. Adelstein RS, and Eisenberg E: Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem 49: 921–956, 1980. 11. Chacko S, Conti MA, and Adelstein RS: Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2⫹ regulation. Proc Natl Acad Sci USA 74: 129 –133, 1977.
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12. Sellers JR, Pato MD, and Adelstein RS: Reversible phosphorylation of smooth muscle myosin, heavy meromyosin, and platelet myosin. J Biol Chem 256: 13137–13142, 1981. 13. Dillon PF, Aksoy MO, Driska SP, et al: Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211: 495– 497, 1981. 14. Kamm KE, and Stull JT: Myosin phosphorylation, force, and maximal shortening velocity in neurally stimulated tracheal smooth muscle. Am J Physiol 249(3 Pt 1): C238 – C247, 1985. 15. Kitazawa T, Masuo M, and Somlyo AP: G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 88: 9307–9310, 1991. 16. Kawano Y, Fukata Y, Oshiro N, et al: Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol 147: 1023–1038, 1999. 17. Kawano Y, Yoshimura T, and Kaibuchi K: Smooth muscle contraction by small GTPase Rho. Nagoya J Med Sci 65: 1– 8, 2002. 18. Hartshorne DJ, and Hirano K: Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. Mol Cell Biochem 190: 79 – 84, 1999. 19. Bing W, Chang S, Hypolite JA, et al: Obstruction-induced changes in urinary bladder smooth muscle contractility: a role for Rho kinase. Am J Physiol Renal Physiol 285: F990 –F997, 2003. 20. Marston SB, and Redwood CS: The molecular anatomy of caldesmon. Biochem J 279: 1–16, 1991. 21. Sobue K, Muramoto Y, Fujita M, et al: Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci USA 78: 5652–5655, 1981. 22. Shirinsky VP, Biryukov KG, Hettasch JM, et al: Inhibition of the relative movement of actin and myosin by caldesmon and calponin. J Biol Chem 267: 15886 –15892, 1992. 23. Dabrowska R, Goch A, Galazkiewicz B, et al: The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta 842: 70 –75, 1985.
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ALTERATION OF CONTRACTILE AND REGULATORY PROTEINS IN ESTROGEN-INDUCED HYPERTROPHY OF FEMALE RABBIT BLADDER ALPHA DIAN-YU LIN, ROBERT M. LEVIN, BARRY A. KOGAN, CATHERINE WHITBECK, ROBERT E. LEGGETT, CHRISTINE KEARNS, AND ANITA MANNIKAROTTU
ABSTRACT Objectives. Estrogen is essential to mediate physiologic functions in female bladders. Deficiency of estrogen has been speculated to be an etiologic factor for bladder dysfunction in postmenopausal women. Our previous studies have demonstrated that estrogen supplementation in female rabbits induces a “functional hypertrophy” of the urinary bladder smooth muscle. The present study investigated the alterations in the contractile and regulatory proteins in this model. Methods. Twenty New Zealand white female rabbits were separated into five groups of 4 rabbits each. Group 1 served as the control, groups 2 to 6 underwent ovariectomy (Ovx), and group 2 served as the Ovx without estradiol treatment group. Two weeks after Ovx, groups 3 to 5 were given 17-beta estradiol (1 mg/kg/day) by subcutaneous implant for 1, 3, and 7 days, respectively. The expression of the contractile and regulatory proteins, such as myosin light chain kinase, rho-kinase, and caldesmon, was analyzed by Western blotting. Results. The expression of myosin light chain kinase was enhanced by estradiol supplementation. The expression of rho-kinase-alpha was increased significantly (20-fold) after Ovx, which was downregulated after estrogen supplementation. No significant change was seen in rho-kinase-beta after Ovx or estradiol supplementation. The expression of caldesmon isoforms was enhanced by 1-day estradiol supplementation but decreased to lower levels than those of the control group by 3 and 7 days of estrogen treatment. Conclusions. The results of the present study have provided more understanding about the role of the contractile and regulatory proteins in detrusor muscle, in both dysfunctional atrophy induced by Ovx and functional hypertrophy induced by estrogen supplementation. UROLOGY 68: 1139–1143, 2006. © 2006 Elsevier Inc.
I
n women, the genital and urinary tract organs are both sensitive to female sex hormones, perhaps because of their common embryologic origin. It has been established that the alterations in circulating estrogen levels that occur during menstrua-
This material was based on work supported in part by the Office of Research and Development (Medical Research Service) of the Department of Veterans Affairs and in part by National Institutes of Health grant RO-1-DK 067114, as well as by the Capital Region Medical Research Foundation. From the Albany College of Pharmacy; Albany Medical College, Albany, New York; Taichung Poh-Ai Hospital, Taichung, Taiwan; and Stratton Veterans Affairs Medical Center, Albany, New York Reprint requests: Anita S. Mannikarottu, Ph.D., Albany College of Pharmacy, 106 New Scotland Avenue, Albany, NY 12208. E-mail: [email protected] Submitted: March 15, 2006, accepted (with revisions): August 22, 2006 © 2006 ELSEVIER INC. ALL RIGHTS RESERVED
tion, pregnancy, and menopause can have marked effects on urogenital organ function.1,2 The postmenopausal estrogenic deficit could be related to many urogenital problems. Even though many investigators have considered menopause to be a major risk factor for incontinence, a direct correlation has never been confirmed.3 However, estrogen has been used clinically for the treatment of urinary incontinence in postmenopausal women.4,5 Alterations of contractile and regulatory proteins have been reported in obstructive bladder in animal models mimicking men with benign prostatic hyperplasia-induced bladder outlet obstruction.6 In response to partial bladder outlet obstruction, the rabbit urinary bladder results, initially, in smooth muscle hypertrophy, increased bladder mass, and remodeling of the bladder wall and contractile dysfunctions. Estrogen supplementation to ovariectomized 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.08.1094 1139
rabbits results in significant bladder hypertrophy of a similar magnitude as that observed after partial outlet obstruction in men.7 Using estradiol supplementation, we had reported on an animal model of estrogen-induced functional hypertrophy.8 In contrast to the dysfunctional hypertrophy of the urinary bladder detrusor smooth muscles induced by bladder outlet obstruction, estrogen supplementation induced an enlarged bladder with greater contractility and an increased smooth muscle/collagen ratio. It is our hypothesis that the contractile and regulatory proteins are altered in the functional hypertrophy model by estrogen supplementation and contribute to the augmented contractility of bladder smooth muscle. The present study investigated the estradiol-induced alterations of those proteins. MATERIAL AND METHODS ANIMALS The Institutional Animal Care and Use Committee of the Stratton Veterans Affairs Medical Center approved this project. Twenty female New Zealand white rabbits weighing 3.5 to 4.0 kg were separated into five groups of 4 rabbits each. Group 1 served as the control group and groups 2 to 5 underwent ovariectomy (Ovx). For Ovx, each rabbit was anesthetized with isoflurane (1% to 3%) and both ovaries were excised through bilateral incisions that were closed with 2-0 silk suture. After 14 days, the group 2 rabbits were killed and the bladders excised. The rabbits in groups 3 to 5 were medicated under anesthesia with 17-beta estradiol (Innovative Research of America, Sarasota, Fla) at a dose of 1 mg/kg/day for 1, 3, and 7 days, respectively, by surgical implantation of an estradiol tablet in the subscapular area.
TISSUE PREPARATION The bladder was excised through a midline incision and opened longitudinally from the base to the dome. Three fullthickness longitudinal strips, about 2 ⫻ 10 mm from the ventral surface, were used for the contractile studies, and the balance of the bladder body was frozen in liquid nitrogen and stored at ⫺80°C for molecular analysis.
CONTRACTILITY STUDIES Each strip was mounted in a 15-mL organ bath containing Tyrode’s solution composed of 124.9 mM NaCl, 2.5 mM KCl, 23.8 mM NaHCO3, 0.5 mM MgCl2, 0.4 mM NaH2PO4, 1.8 mM CaCl2, and 5.5 mM dextrose at 37°C for 2 hours. The buffer was equilibrated with a mixture of 95% oxygen and 5% carbon dioxide. Tension was monitored using a Model D Grass Polygraph and digitized using the Grass Polyview system. Preliminary length-tension studies demonstrated that maximal active tension was obtained at 2 g of passive tension for the control and experimental bladders. The strips were equilibrated for 30 minutes at 2-g resting tension. They were then stimulated at 2, 8, and 32 Hz for 20 seconds with pulses 1 ms in duration at 80 V every 5 minutes. Next, the responses to 1 mM adenosine triphosphate (ATP), 20 M carbachol, and 120 mM KCl were determined. Between the additions of the pharmacologic agents, each tissue strip was washed three times at 15-minute intervals with fresh buffer. 1140
PROTEIN PREPARATION, SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS, AND WESTERN BLOTTING Frozen bladder tissues (100 mg) were pulverized while immersed in liquid nitrogen using a mortar and homogenized in buffer containing 20% glycerol, 50 mM tris-HCl (pH 6.8), 0.5% (vol/vol) Tween-20, and protease inhibitors (0.5 mM phenylmethanesulfonyl fluoride, 2 M pepstatin, 2 M antipain, and 0.1 mg/mL trypsin inhibitor).The homogenate was centrifuged at 38,000g for 15 minutes using a centrifuge (Beckman, GS-15R), and the supernatant was collected. After gently mixing the samples in sodium dodecyl sulfate (final concentration 1%), they were boiled for 4 minutes and centrifuged at 10,000 rpm for 15 minutes. The protein concentration was determined using the DC protein assay kit (BioRad Laboratories, Hercules, Calif). Equal amounts (20 g) of total protein from each group were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (except for caldesmon [CaD], which was in 7.5% gel) and transferred to Immobilon-P membranes with Towbin buffer (25 mM tris, 192 mM glycine, and 20% methanol). The membranes were blocked with 5% nonfat milk in 0.05% Tween-20 in phosphate-buffered saline for 1 hour at 37°C and then incubated with primary antibody, myosin light chain kinase (MLCK, 1:2000), rhokinase (ROK)-alpha (clone 21, (1:500), and ROK-beta (clone 46, 1:250, all three from BD Biosciences, San Jose, Calif), CaD (clone C21; 1:1000, Sigma-Aldrich, St. Louis, Mo), in a shaking incubator. The membranes were washed with buffer (20 mM tris, 500 mM NaCl, and 0.05% Tween 20) and incubated with secondary antibody (horseradish peroxidase-conjugated goat antimouse IgG at 1:10,000) for 1 hour at 37°C. Substrates were visualized by echo-chemiluminescence. Band intensities were scanned and analyzed with a Kodak Image Station 440CF and Kodak ID image analysis software (Scientific Image System, Rochester, NY).
STATISTICAL ANALYSIS The data were analyzed using analysis of variance followed by the Neuman-Keuls test for individual differences using Sigma Stat, version 2.03 (SPSS, Chicago, Illinois). P ⬍0.05 was considered statistically significant.
RESULTS The bladders from the Ovx group showed decreased responses to all forms of stimulation (field stimulation, carbachol, and KCl) compared with those from the control group. At 7 days of estrogen treatment, the contractile responses had increased significantly compared with those in the Ovx group and equaled those of the control group (Fig. 1). Figure 2 shows that the expression of MLCK was enhanced by 1, 3, and 7-day estradiol supplementation. Figure 3 demonstrates that the expression of ROK-alpha increased significantly (20-fold) after Ovx and was downregulated after estrogen supplementation. No significant change was seen in ROK-beta after Ovx or estradiol supplementation (Fig. 4). Figure 5 shows that CaD isoforms (heavy chain and light chain) dysregulated at Ovx, enhanced by 1-day estradiol supplementation, and then decreased to lower levels than in the control group with 3 and 7 days of estrogen treatment. UROLOGY 68 (5), 2006
FIGURE 1. Contractile responses to FS, ATP, carbachol, and KCl. Ovx group showed decreased responses to all stimulations compared with control group. In 7-day group, significantly increased responses were noted after 7 days of estradiol treatment compared with Ovx group. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05.
FIGURE 2. (A) Western blot analyses of MLCK expression in each group. Equal amounts of total extractable proteins (20 g) from control, Ovx, and estrogentreated rabbit bladder smooth muscles were separated by electrophoresis, transferred to membrane, and probed with antibody specific to MLCK, as described. Lane 1, control (normal female rabbit); lane 2, ovariectomized for 2 weeks; lanes 3, 4, and 5, estrogen treated for 1, 3, and 7 days, respectively. Ovx group did not show any significant change in MLCK expression. Note, overexpression of MLCK in estrogentreated groups. (B) Average expression of MLCK in different samples. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05. UROLOGY 68 (5), 2006
FIGURE 3. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to ROK-alpha. Note, 20-fold increase in ROK-alpha expression in Ovx rabbits. (B) Average expression of ROKalpha in different samples. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group; xsignificantly different from Ovx group, P ⬍0.05.
FIGURE 4. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to ROK-beta in each group. No significant change was seen in expression of ROK-beta in different groups. (B) Average expression of ROK-beta in each group. Each bar represents mean ⫾ SEM for n ⫽ 4.
COMMENT Myosin II is the molecular motor for contraction in all muscle types. In general, the velocity of force generation by the muscle is determined by the ATPase activity of myosin II when it interacts with 1141
FIGURE 5. (A) Representative Western blot of bladder tissue homogenates probed with antibody specific to CaD in each group. (B) Average expression of CaD isoforms, h-CaD and l-CaD, in each group. Each bar represents mean ⫾ SEM for n ⫽ 4. *Significantly different from control group, P ⬍0.05.
actin.9 Myosin II in smooth muscle cells is activated by actin when the regulatory light chains (MLC20) are phosphorylated by a Ca2⫹-calmodulin-dependent MLCK.10,11 Dephosphorylation of the MLC20 by Ca2⫹-independent phosphatase (MLCP) lowers the actin-activated ATPase activity of myosin.12 A correlation between myosin phosphorylation-dephosphorylation and contractionrelaxation of the smooth muscle has been previously demonstrated.13,14 Myosin phosphorylation and subsequent force can be maintained by inhibiting MLCP.15 Our study demonstrated that Ovx resulted in decreased bladder contractile function and that the contractility returned to normal after estradiol supplementation. However, the present results revealed that MLCK expression was enhanced by 1, 3, and 7-day estradiol supplementation. Estrogen might have enhanced the MLCK activity so as to upregulate MLC phosphorylation, which increased the following contractile response. MLCP is regulated by the small guanosine triphosphatase RhoA and Rho-associated kinase.16 The inhibition of MLCP by RhoA/ROK is associated with the phosphorylation of the MLCP and with smooth muscle contraction in the absence of a rise in intracellular Ca2⫹ concentrations.17 Thus, ROK-mediated regulation of MLC phosphorylation increases the Ca2⫹ sensitivity of smooth muscle contraction.18 An enhanced RhoA/ROK-mediated signal transduction pathway (ROK-alpha) was found in the Ovx and estradiol-augmented groups. The involvement of this pathway suggests a remodeling in the regulation of myosin phos1142
phorylation, which in turn regulates the actin-myosin interaction and contraction in detrusor muscles. Ovx-induced ROK-alpha overexpression might indicate the compensatory effect of the bladder in response to the Ovx and hypoxic damage, despite the failure to increase contractility. Estrogen-induced ROK-alpha overexpression might indicate the promoted myosin phosphorylation, which in turn upregulates the actin-myosin interaction and contraction in detrusor muscles. In contrast to partial bladder outlet obstruction-induced bladder dysfunction, in which the expression of ROK-beta was increased,19 no significant change was found in the ROK-beta expression among the control, Ovx, and estradiol-supplemented groups in the present study. The actin-associated protein CaD is thought to modulate smooth muscle contraction, regulated through thick myosin filaments by way of phosphorylation of the MLC.20 CaD is expressed as two dominant isoforms, l-CaD and h-CaD (low- and high-molecular sizes, respectively). l-CaD is found in nonmuscle cells of various tissue types and h-CaD is present in smooth muscle cells from all sources.21 h-CaD is bound to thin filaments in smooth muscle where it inhibits the actin-myosin interaction and actomyosin ATPase activity. h-CaD inhibits the in vitro motility of actin filaments over myosin heads.22 The role of l-CaD in contractility or cell motility is less clear, although it has been shown to dissociate actin bundles in the presence of Ca2⫹.23 Ovx induced dysregulation in the expression of CaD isoforms, with greater l-CaD and lower expression of h-CaD compared with the control group. The overexpressed l-CaD may be associated with remodeling of the cytoskeletal structure and that the overexpressed l-CaD displaces h-CaD from the thin filaments and that thin filaments containing l-CaD may interfere with the generation and/or maintenance of the additional force required for compensating the Ovx-induced contractile dysfunction. The expressions of both isoforms of CaD were significantly decreased in the estrogen treatment groups by 3 and 7 days, which may have contributed to the increased contractility seen in those groups. The stabilized equilibrium, which was lower than in the control group, might be associated with the augmented contractile function. In contrast to the obstructed bladder model, the expression of l-CaD and h-CaD in the estrogeninduced functional hypertrophy bladder was downregulated, with a certain stable ratio compared with the normal control bladder. CONCLUSIONS The results of this study have demonstrated estrogen-augmented functional hypertrophy and alterations in the contractile and regulatory proteins. UROLOGY 68 (5), 2006
Future investigation of these pathways might provide more understanding of the proteins and the associated modulations. Targeting the associated pathways in the regulation of detrusor muscle contraction would be interesting. REFERENCES 1. Batra SC, and Iosif CS: Female urethra: a target for estrogen action. J Urol 129: 418 – 420, 1983. 2. McGuire EJ: Pathophysiology of incontinence in elderly women, in O’Donnell PD (Ed): Geriatric Medicine. New York, Little, Brown, 1994, pp 221–228. 3. Tinelli A, Tinelli R, Perrone A, et al: Urinary incontinence in postmenopausal period: clinical and pharmacological treatments. Minerva Ginecol 57: 593– 609, 2005. 4. Faber P, and Heidenreich J: Treatment of stress incontinence with estrogen in postmenopausal women. Urol Int 32: 221–227, 1977. 5. Fantl JA, Wyman JF, and Anderson LR: Postmenopausal urinary incontinence: comparison between nonestrogen-supplemented and estrogen-supplemented woman. Obstet Gynecol 71: 823– 828, 1988. 6. Chacko S, Chang S, Hypolite J, et al: Alteration of contractile and regulatory proteins following partial bladder outlet obstruction. Scand J Urol Nephrol Suppl 215: 26 –36, 2004. 7. Aikawa K, Sugino T, Matsumoto S, et al: The effect of ovariectomy and estradiol on rabbit bladder smooth muscle contraction and morphology. J Urol 170(2 Pt 1): 634 – 637, 2003. 8. Lin AD, Levin R, Kogan B, et al: Estrogen induced hypertrophy is accompanied by increased force generation. Neurourol Urodyn May 10, 2006; Epub ahead of print. 9. Barany M: ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50(suppl 218): 197–218, 1967. 10. Adelstein RS, and Eisenberg E: Regulation and kinetics of the actin-myosin-ATP interaction. Annu Rev Biochem 49: 921–956, 1980. 11. Chacko S, Conti MA, and Adelstein RS: Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2⫹ regulation. Proc Natl Acad Sci USA 74: 129 –133, 1977.
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