Hydrogen sulfide has a role as an endogenous relaxation factor in the bladder and prostate of male rats

Shimizu T1, Zou S1, Shimizu S1, Higashi Y1, Nakamura K1, Ono H1, Aratake T1, Yamamoto M1, Honda M2, Saito M1

Research Type

Basic Science / Translational

Abstract Category

Pharmacology

Abstract 50
Basic Science: Pharmacology
Scientific Podium Short Oral Session 5
Wednesday 29th August 2018
11:22 - 11:30
Hall B
Animal Study Basic Science Male Pharmacology
1. Department of Pharmacology, Kochi Medical School, Kochi University, Nankoku, Japan, 2. Division of Urology, Tottori University School of Medicine, Yonago, Japan
Presenter
T

Takahiro Shimizu

Links

Abstract

Hypothesis / aims of study
In the lower urinary tract, donors of hydrogen sulfide (H2S), an endogenous gasotransmitter [1], induce contraction of the rat detrusor [2] and relaxation of the pig bladder neck [3].  These reports suggest a possibility that H2S may have site-specific effects on the bladder.  However, the detailed functions of H2S in each part of the bladder are still unclear.  In addition, there are no reports showing physiological roles of H2S in the prostate.
H2S biosynthesis is mediated by three enzymatic mechanisms: two responsible enzymes for metabolism of L-cysteine, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), and the third mechanism, the combined action of 3-mercaptopyruvate sulfurtransferase (MPST) and cysteine aminotransferase (CAT) [1].  CBS and CSE produce H2S from L-cysteine and L-homocysteine.  MPST produces H2S from 3-mercaptopyruvate, which is synthesized from L-cysteine by CAT (MPST/CAT pathway).  Recently, D-cysteine can be also a significant substrate for H2S production; it is converted to 3-mercaptopyruvate by D-amino acid oxidase (DAO) (MPST/DAO pathway) [1].
In this study, we investigated (1) tissue distributions of CBS, CSE, MPST, CAT and DAO, (2) endogenous H2S levels, and (3) effects of H2S donors (NaHS and GYY4137) on contractility, in the rat bladder and prostate.
Study design, materials and methods
Bladder dome and trigone (BL-D and BL-T), dorsolateral and ventral prostate (PR-D and PR-V), liver and cerebellum (L and C) were prepared from male Wistar rats (300-400 g) sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip).
(1) Tissue distributions of 5 enzymes were investigated by real-time PCR, Western blot and immunohistochemistry.  L and C were used for positive controls for real-time PCR, Western blot and immunohistochemistry.  (2) H2S contents in the bladder and prostate tissues were measured by methylene blue method.  (3) By using 1 x 5 mm strips of the bladder and prostate tissues, effects of NaHS (1 x 10-9  to 3 x 10-4  M) were evaluated on pre-contracted bladder strips by carbachol (10-5  M) and prostate strips by noradrenaline (10-5  M).  Prostate strips were pretreated with propranolol (10-6  M) 30 min before the pre-contraction.
In addition, in vivo study was also performed.  In urethane anesthetized (0.8 g/kg, ip) male Wistar rats (350-400 g), a catheter was inserted into the bladder in order to instill reagents (2.4 ml/h) and to measure intravesical pressure.  After detecting 4-5 micturition reflexes induced by saline instillation, GYY4137 solution (10-8, 10-7, and 10-6 M) or vehicle was instilled.
Results
(1) MPST and CAT were detected in the bladder and prostate and CBS was only
detected in the prostate (Fig. 1A).  Expression levels of MPST in the prostate were higher than those in the bladder, and those in the PR-D were higher than those in the PR-V (Fig. 1A).  In expression levels of CAT, there was no significant difference among each tissue (Fig. 1A).  Expression levels of CBS in the PR-V were higher than those in the PR-D (Fig. 1A).  On the other hand, CSE and DAO were not detected in both tissues (data not shown).  Immunoreactvities of these enzymes were mainly detected in the urothelium and smooth muscle layer of the bladder, and in the prostate glandular epithelium (Fig. 1B).
(2) H2S was detected in the bladder and prostate tissues (Fig. 1C).
(3) NaHS dose-dependently induced relaxation of pre-contracted BL-D, BL-T, PR-D and PR-V strips.  There were no significant differences of the EC50 values or maximal relaxation rate against the pre-contractions among any of these tissues (Table 1A).
Intravesically instilled GYY4137 significantly prolonged intercontraction intervals (ICI) compared to the vehicle-treated group (Table 1B), and showed, although non-significant, tendency to reduce maximal voiding pressure (MVP) compared to the vehicle-treated group (Table 1B).
Interpretation of results
In the bladder, the MPST/CAT pathway is major for H2S biosynthesis, while in the prostate, CBS and MPST/CAT pathways are involved in the biosynthesis.  Considering tissue distribution of these enzymes, H2S might be produced from smooth muscle layer and urothelium in the bladder, thereby inducing relaxation of detrusor smooth muscle by autocrine and paracrine manners.  In the prostate, these enzymes are mainly distributed in the glandular epithelium, indicating that endogenously produced H2S might relax prostate sommth muscle in a paracrine manner.  In this study, we found endogenous H2S at lower nmol range in homogenized rat bladder and prostate for the first time by the methylene blue method, which is performed in acidic condition; therefore, a limitation of the method is that the incorporation of acid-labile sulfide may impact on the interpretation of the actual H2S level.   Because reliable methods to measure endogenous H2S are lacking, further studies are necessary to examine “more precise” endogenous H2S levels in the bladder and prostate.
Although site-specific effects of H2S donors in the bladder are speculated [2,3], both BL-D and BL-T strips were relaxed by NaHS in this study.  It is reported that NaHS induced contraction of detrusor strips at millimolar orders [2], much higher than those induced bladder strips relaxation in this study.  In addition, intravesically instilled GYY4137 induced prolongation of ICI and reduction of MVP at sub-micromolar doses.  Because gasotransmitters-induced responses are generally much dependent on doses (physiological responses at lower doses and toxic effects at higher doses), physiclogical roles of H2S in the bladder may be an endogenous relaxation factor.  Furthermore, to our knowledge, this is the first report showing H2S-induced relaxation of prostate smooth muscle.
Concluding message
Our present data suggest that H2S has a role as an endogenous relaxation factor in the rat bladder and prostate.  Therefore, endogenous H2S might open new avenues of therapeutic interventions for lower urinary tract dysfunctions such as overactive bladder and benign prostatic hyperplasia.
Figure 1
Figure 2
References
  1. Kimura H. The physiological role of hydrogen sulfide and beyond. Nitric Oxide. 2014;41:4-10.
  2. Patacchini R, Santicioli P, Giuliani S, et al. Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle. Eur J Pharmacol. 2005;509:171-177.
  3. Fernandes VS, Ribeiro AS, Martínez MP, et al. Endogenous hydrogen sulfide has a powerful role in inhibitory neurotransmission to the pig bladder neck. J Urol. 2013;189:1567-1573.
Disclosures
<span class="text-strong">Funding</span> JSPS KAKENHI Grant (#15K15583), GSK Japan Research Grant. <span class="text-strong">Clinical Trial</span> No <span class="text-strong">Subjects</span> Animal <span class="text-strong">Species</span> Rat <span class="text-strong">Ethics Committee</span> The Kochi University Institutional Animal Care and Use Committee