Korean J Urol.  2014 Oct;55(10):677-686. 10.4111/kju.2014.55.10.677.

Expression of alpha1 Receptor and Nitric Oxide Synthase in Oophorectomized and Estrogen-Supplemented Rat Bladder and Urethra

• Seo Y
• Park SW
• Kim JY
• Lee SD

• KMID: 2069792
• DOI: http://doi.org/10.4111/kju.2014.55.10.677

Abstract

PURPOSE
To investigate the effects of estrogen on the expression of the alpha1 receptor and nitric oxide synthase (NOS) in rat urethra and bladder after oophorectomy.
MATERIALS AND METHODS
Forty-five mature female Sprague-Dawley rats (aged 10-11 weeks, 235-250 g) were randomly assigned to one of three groups: control group, oophorectomy group (Opx), or oophorectomy and estradiol replacement group (Opx+ Est). The degree of expression of alpha1 receptor (alpha1A and D) and NOS (neuronal NOS [nNOS] and endothelial NOS [eNOS]) in bladder and urethral tissues was investigated by using immunohistochemical staining and Western blotting.
RESULTS
In the bladder, the expression rates of alpha1 receptor (alpha1A and alpha1D) increased in the Opx group but decreased in the Opx+Est group. These changes were not statistically significant. The alpha1A and alpha1D receptor of the urethra decreased in the Opx group but increased in the Opx+Est group. These changes were not statistically significant. In the bladder and urethra, the expression rates of nNOS and eNOS significantly increased in the Opx group but decreased in the Opx+Est group (p<0.05).
CONCLUSIONS
These data suggest that estrogen depletion increases NOS and alpha1 receptor expression in the rat bladder. However, these changes could be restored by estrogen replacement therapy.

Keyword

Bladder; Estrogens; Nitric oxide synthase; Urethra

MeSH Terms

Animals
Collagen/metabolism
Estradiol/analogs & derivatives/blood/pharmacology
Estrogen Replacement Therapy/*methods
Female
Muscle, Smooth/pathology
Nitric Oxide Synthase/*metabolism
Ovariectomy
Rats, Sprague-Dawley
Receptors, Adrenergic, alpha-1/*metabolism
Urethra/drug effects/*metabolism/pathology
Urinary Bladder/drug effects/*metabolism/pathology
Collagen
Estradiol
Nitric Oxide Synthase
Receptors, Adrenergic, alpha-1

Figure

• 

  FIG. 1 α1A receptor protein expression in the rat bladder. (A) Protein is extracted from the bladder in the Con, Opx, and Opx+Est group, and it is analyzed for the α1A receptor and actin levels by Western blotting. (B) The relative amount of α1A receptor protein increased after oophorectomy. It is normalized after Estradiol replacement. However, the expressions of α1A receptor protein in the Con, Opx, and Opx+Est group are not significantly different. Con, control; Opx, oophorectomy; Opx+ Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 2 α1D receptor protein expression in the rat bladder. (A) Protein is extracted from the bladder in the Con, Opx, and Opx+Est group, and it is analyzed for the α1D receptor and actin levels by Western blotting. (B) The relative amount of α1D receptor protein increased after oophorectomy. It is normalized after Estradiol replacement. However, the expressions of α1D receptor protein in the Con, Opx, and Opx+Est group are not significantly different. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 3 Immunreactivity pattern for neuronal nitric oxide synthase (nNOS) of rat bladder (immunohistochemical stain, ×400). The blood vessel, the expression of nNOS increased in the Opx group in comparison to the Con group (A), and decreased in the Opx+Est group (B) in comparison to the Opx group (C). Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 4 Neuronal nitric oxide synthase (nNOS) protein expression in the rat bladder. (A) Protein is extracted from the bladder in the Con, Opx, and Opx+Est group, and it is analyzed for the nNOS and actin levels by Western blotting. (B) The relative amount of nNOS protein significantly increased after oophorectomy. It is normalized after Estradiol replacement. *p<0.05. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 5 Immunoreactivity pattern for endothelial nitric oxide synthase (eNOS) of rat bladder (immunohistochemical stain, ×400). The expression of eNOS increased in Opx group in comparison to the Con group (A), while it decreased in the Opx+Est group (B) in comparison to the Opx group (C). Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 6 Endothelial nitric oxide synthase (eNOS) protein expression in the rat bladder. (A) Protein is extracted from the bladder in the Con, Opx, and Opx+Est group, and it is analyzed for the eNOS and actin levels by Western blotting. (B) The relative amount of eNOS protein significantly increased after oophorectomy. It is normalized after Estradiol replacement. *p<0.05. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 7 Masson's trichrome staining of the rat bladder (Masson's trichrome staining, ×200). The proliferation of epithelial cells and the increase in the muscle layer of the bladder were observed for the control (Con) group (A), Opx+Est group (B), Opx group (C). Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 8 α1A receptor protein expression in the rat urethra. (A) Protein is extracted from the urethra in the Con, Opx, and Opx+Est group, and it is analyzed for the α1A receptor and actin levels by Western blotting. (B) The relative amount of α1A receptor protein decreased after oophorectomy. It is normalized after Estradiol replacement. However, the expressions of α1A receptor protein in the Con, Opx, and Opx+Est group are not significantly different. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 9 α1D receptor protein expression in the rat urethra. (A) Protein is extracted from the urethra in the Con, Opx, and Opx+Est group, and it is analyzed for the α1D receptor and actin levels by Western blotting. (B) The relative amount of α1D receptor protein increased after oophorectomy. It is normalized after Estradiol replacement. However, the expressions of α1D receptor protein in the Con, Opx, and Opx+Est group are not significantly different. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 10 Immunreactivity pattern for endothelial nitric oxide synthase (NOS) of rat urethra (immunohistochemical stain, ×400). The expression of neuronal NOS increased in all of the mucous membrane, smooth muscle and blood vessel of the urethra in the Opx group (B). (A) Con, control; (B) Opx, oophorectomy. (C) Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 11 Neuronal nitric oxide synthase (nNOS) protein expression in the rat urethra. (A) Protein is extracted from the urethra in the Con, Opx, and Opx+Est group, and it is analyzed for the nNOS and actin levels by Western blotting. (B) The relative amount of nNOS protein significantly increased after oophorectomy. It is normalized after Estradiol replacement. *p<0.05. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 12 Immunoreactivity pattern for endothelial nitric oxide synthase (eNOS) of rat urethra (immunohistochemical stain, ×400). The expression of eNOS increased in Opx group (B) in comparison to the Con group (A), while it decreased in the Opx+Est group (C) in comparison to the Opx group. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 13 Endothelial nitric oxide synthase (eNOS) protein expression in the rat urethra. (A) Protein is extracted from the urethra in the Con, Opx, and Opx+Est group, and it is analyzed for the eNOS and actin levels by Western blotting. (B) The relative amount of eNOS protein significantly increased after oophorectomy. It is normalized after Estradiol replacement. *p<0.05. Con, control; Opx, oophorectomy; Opx+Est, 17β-estradiol replacement after oophrorectomy.

• 

  FIG. 14 Masson's trichrome staining of the rat urethra (Masson's trichrome staining, ×200). The increase in the distribution of blood vessels, proliferation of epithelial cells and the increase in the muscle layer of the urethra were observed for the Opx+Est group (C). (A) Con, control; (B) Opx, oophorectomy. (C) Opx+Est, 17β-estradiol replacement after oophrorectomy.

Reference

1. Takahashi W, Yoshida M, Wada Y, Goto S, Inadome A, Yono M, et al. Effect of estrogen on nitric oxide-induced relaxation of the rabbit urethra. Eur J Pharmacol. 1997; 339:165–171.

2. Morrison JF. The functions of efferent nerves to the lower urinary tract. In : Torrens M, Morrison JF, editors. The physiology of the lower urinary tract. London: Springer;1987. p. 133–159.

3. Seguchi H, Nishimura J, Zhou Y, Niiro N, Kumazawa J, Kanaide H. Expression of beta3-adrenoceptors in rat detrusor smooth muscle. J Urol. 1998; 159:2197–2201.

4. Maggi CA, Santicioli P, Furio M, Meli A. Dual effects of clonidine on micturition reflex in urethane anesthetized rats. J Pharmacol Exp Ther. 1985; 235:528–536.

5. Hampel C, Dolber PC, Smith MP, Savic SL, Throff JW, Thor KB, et al. Modulation of bladder alpha1-adrenergic receptor subtype expression by bladder outlet obstruction. J Urol. 2002; 167:1513–1521.

6. Dokita S, Morgan WR, Wheeler MA, Yoshida M, Latifpour J, Weiss RM. NG-nitro-L-arginine inhibits non-adrenergic, noncholinergic relaxation in rabbit urethral smooth muscle. Life Sci. 1991; 48:2429–2436.

7. Hashimoto S, Kigoshi S, Muramatsu I. Nitric oxide-dependent and -independent neurogenic relaxation of isolated dog urethra. Eur J Pharmacol. 1993; 231:209–214.

8. Leone AM, Wiklund NP, Hokfelt T, Brundin L, Moncada S. Release of nitric oxide by nerve stimulation in the human urogenital tract. Neuroreport. 1994; 5:733–736.

9. Yallampalli C, Byam-Smith M, Nelson SO, Garfield RE. Steroid hormones modulate the production of nitric oxide and cGMP in the rat uterus. Endocrinology. 1994; 134:1971–1974.

10. Jackson SL, Fihn SD. Exogenous estrogen and urinary incontinence. J Urol. 2009; 181:1989–1991.

11. Chen B, Yeh J. Alterations in connective tissue metabolism in stress incontinence and prolapse. J Urol. 2011; 186:1768–1772.

12. Hextall A, Cardozo L. The role of estrogen supplementation in lower urinary tract dysfunction. Int Urogynecol J Pelvic Floor Dysfunct. 2001; 12:258–261.

13. Grady D, Brown JS, Vittinghoff E, Applegate W, Varner E, Snyder T, et al. Postmenopausal hormones and incontinence: the Heart and Estrogen/Progestin Replacement Study. Obstet Gynecol. 2001; 97:116–120.

14. Moehrer B, Hextall A, Jackson S. Oestrogens for urinary incontinence in women. Cochrane Database Syst Rev. 2003; (2):CD001405.

15. Beisland HO, Fossberg E, Moer A, Sander S. Urethral sphincteric insufficiency in postmenopausal females: treatment with phenylpropanolamine and estriol separately and in combination. A urodynamic and clinical evaluation. Urol Int. 1984; 39:211–216.

16. Dmitrieva N. Increased alpha1-adrenergic activity in the rat bladder by depletion of ovarian hormones. J Urol. 2007; 178:2677–2682.

17. Rand MJ, Li CG. Discrimination by the NO-trapping agent, carboxy-PTIO, between NO and the nitrergic transmitter but not between NO and EDRF. Br J Pharmacol. 1995; 116:1906–1910.

18. Bennett BC, Kruse MN, Roppolo JR, Flood HD, Fraser M, de Groat WC. Neural control of urethral outlet activity in vivo: role of nitric oxide. J Urol. 1995; 153:2004–2009.

19. Ho MH, Bhatia NN, Khorram O. Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract. Curr Opin Obstet Gynecol. 2004; 16:423–429.

20. Okuno T, Masuda H, Tsujii T, Kihara K, Yamauchi Y, Azuma H. Accumulated endogenous nitric oxide synthase inhibitors in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits. J Urol. 2004; 172:360–364.