Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling

Abstract

Testosterone (T) plays a permissive role in the development of benign prostatic hyperplasia (BPH), and phosphodiesterase 5 inhibitors (PDE5is) have been found to be effective for BPH and lower urinary tract symptoms (LUTS) in clinical trials. This study investigated the effect of T on smooth muscle (SM) contractile and regulatory signaling pathways, including PDE5 expression and functional activity in prostate in male rats (sham-operated, surgically castrated, and castrated with T supplementation). In vitro organ bath studies, real-time RT-PCR, Western blot analysis, and immunohistochemistry were performed. Castration heavily attenuated contractility, including sensitivity to phenylephrine with SM myosin immunostaining revealing a disrupted SM cell arrangement in the stroma. PDE5 was immunolocalized exclusively in the prostate stroma, and orchiectomy signficantly reduced PDE5 immunopositivity, mRNA, and protein expression, along with nNOS and ROKβ mRNA, whereas it increased eNOS plus α(1a) and α(1b) adrenoreceptor expression in castrated animals. The PDE5i zaprinast significantly increased prostate strip relaxation to the nitric oxide donor sodium nitroprusside (SNP) in control but not castrated rats. But SNP alone was more effective on castrated rats, comparable with sham treated with SNP plus zaprinast. T supplementation prevented or restored all above changes, including SNP and zaprinast in vitro responsiveness. In conclusion, our data show that T positively regulates PDE5 expression and functional activities in prostate, and T ablation not only suppresses prostate size but also reduces prostatic SM contractility, with several potential SM contraction/relaxation pathways implicated. Zaprinast findings strongly suggest a major role for PDE5/cGMP in this signaling cascade. PDE5 inhibition may represent a novel mechanism for treatment of BPH.

Fig. 1.

Effect of testosterone (T) on body weight, ventral prostate weight, and seminal vesicle weight. A: bar graph of the average initial and final body weights of all rats used. B: photograph of a typical ventral prostate from a sham (a), castrated (b), and castrated + T rat (c). C: bar graph of the average weight of the ventral prostates from all experimental groups. D: bar graph of the average weight of the seminal vesicles from all experimental groups. **P < 0.001 vs. castration (n = total of 46 rats).

Fig. 2.

Hematoxylin and eosin (H & E) staining and immunolocalization of smooth muscle (SM) myosin of ventral prostate. Representative sections of rat ventral prostate from sham (left), castrated (middle), and castrated + T (right). Top: H & E sections with all magnification ×100. Bottom: immunodistribution of SM myosin in prostatic stroma (magnification ×400). Black arrows indicate intense SM mysoin heavy chain staining in the stroma, whereas weak cross-reactivity is noted in the epithelium.

Fig. 3.

Immunolocalization of phosphodiesterase 5 (PDE5) in rat ventral prostate. A, top: PDE5 positive immunoreactivity in the prostatic stroma from sham-operated rats with magnification ×100 (left) and ×400 (right). A, bottom left: positive PDE5 control (rat lung, magnification ×400); A, bottom right: negative IgG control staining (rat lung, magnification ×400). B: PDE5 immunopositivity in the prostatic stroma from sham (top left), castrated (top right), and castrated + T (bottom left) rats with magnification ×400.

Fig. 4.

Rat ventral prostate SM in vitro contractility. A: summary graph of KCl-induced contraction. Responses to KCl were normalized to strip weight. Values are expressed as means ± SE (n = strips obtained from 46 different animals). B: curves of phenylephrine (PE)-induced dose response contraction. Responses to PE were normalized to strip weight. Values are expressed as means ± SE (n = strips obtained from 46 different animals). **P < 0.01 vs. castration.

Fig. 5.

Expression of PDE5 and other major SM contractility-associated molecules in rat ventral prostrate. The expression of all studied molecules was quantified by real-time RT-PCR. Gene expression was normalized to expression of the ribosomal protein L19 (RPL19) housekeeping gene. PDE5 expression was also determined by Western blot. A: PDE5 mRNA level. *P < 0.05 vs. castration (n = 4–8 different rats for each group). B: PDE5 protein level. Top: representative Western blot bands. Two specific major bands of the expected sizes (85 and 95 kDa) are evident in all lanes. Bottom: densitometric evaluation of PDE5 protein expression, calculated as %band intensity over the control, taken as 100%. *P < 0.05 vs. castration (n = 10 different rats for each group). C: neuronal (nNOS) and endothelial nitric oxide synthase (eNOS) mRNA level. *P < 0.05 vs. sham or castration + T; **P < 0.01 vs. sham or castration + T (n = 4–8 different rats for each group). D: α₁-adrenergic receptor mRNA level. *P < 0.05 vs. sham or castration + T (n = 4–8 different rats for each group). E: Rho kinase (ROK)α and ROKβ mRNA level. *P < 0.05 vs. sham or castration + T (n = 4–8 different rats for each group).

Fig. 6.

PDE5 inhibitor zaprinast potentiating effect on sodium nitroprusside (SNP) in relaxing prostatic SM. Prostate strips were precontracted with 10 μM PE and allowed to reach a stable tension and then relaxed by increasing doses (10⁻⁸–10⁻⁴ M) of SNP with or without 20 nM zaprinast preincubaution. The maximal response to PE was taken as 100%, whereas the relaxant effect of SNP with or without zaprinast was evaluated as a percentage of this response. Values are expressed as means ± SE. *P < 0.05 vs. sham or castration + T; **P < 0.01 vs. sham; ^oP < 0.05 vs. sham or castration + T (n = 4–7 different animals).