Hydrogen sulfide (H2S), an endogenous gasotransmitter, has a wide range of physiological functions including neuromodulation, vasorelaxation and cytoprotection [1].  H2S is endogenously produced by three enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST).  CBS and CSE produce H2S from L-cysteine, while MPST produces H2S from 3-mercaptopyruvate, which is synthesized from L-cysteine by cysteine aminotransferase (CAT) (CAT/MPST pathway) [1].  We recently reported that exogenous H2S induced relaxation of pre-contracted male rat bladder dome (BL-D) and trigone (BL-T) tissue strips and intravesically instilled an H2S donor prolonged intercontraction intervals (ICI) in rats [2].  We also detected that at least the CAT/MPST pathway-mediated endogenous H2S production was working in the rat BL-D and BL-T tissues [2].  These results indicate a possibility that H2S can function as an endogenous relaxation factor in the rat bladder.
Downregulation of the endogenous H2S system is reported in spontaneously hypertensive rats (SHRs), which develop overactive bladder (OAB)-like bladder dysfunction due to hypertension-mediated chronic bladder ischemia [3].  However, hypertension-related changes of the H2S system in the bladder have not been clarified yet.  In this study, therefore, we compared effects of H2S donors (GYY4137 and NaHS) on the micturition reflex and on the bladder contractility, and the endogenous H2S system in the bladder between SHRs and normotensive Wistar rats.

Eighteen-week-old male normotensive Wistar rats and SHRs were used.
(1) Under urethane anesthesia (0.8 g/kg, ip), a catheter was inserted into the bladder from the dome 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.
(2) BL-D and BL-T were prepared from these rats sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip).  By using 1 x 5 mm strips of the bladder, effects of NaHS (1 x 10-8 to 3 x 10-4 M) were evaluated on pre-contracted bladder strips by carbachol (10-5 M).  Tissue H2S content was measured by the methylene blue method.  Expression levels of CBS, CSE, CAT and MPST in the bladder tissues were investigated by Western blot.

(1) The baseline values of ICI (sec, means ± SEM) just before intravesical instillation of vehicle or GYY4137 were 2152±153 in Wistar rats (N=17) and 1362±140 in SHRs (N=14).  The values of SHRs were significantly shorter than those of Wistar rats (P<0.05).  GYY4137 significantly prolonged ICI compared to the vehicle-treated group in Wistar rats, but not in SHRs (Table 1A).
(2) NaHS-induced relaxation on pre-contracted BL-D and BL-T strips was significantly attenuated in SHRs compared with Wistar rats (Table 1B).  The H2S content in the bladder of SHRs was significantly higher than that of Wistar rats (Fig. 1A).  CBS, MPST and CAT, but not CSE, were detected in the bladder of Wistar rats and SHRs (Fig. 1B).  The expression levels of MPST in the SHR bladder were significantly higher than those in the Wistar rat bladder (Fig. 1B).

The difference in basal values of ICI between normotensive Wistar rats and SHRs indicates that SHRs showed frequent urination, in line with previous reports [3].  Intravesically instilled GYY4137 prolonged ICI in Wistar rats and NaHS relaxed pre-contracted bladder strips of Wistar rats, in line with our previous report [2].  These results suggest that intravesically instilled GYY4137-induced inhibition of the micturition reflex in Wistar rats might be mediated at least by relaxation of the bladder smooth muscle.  On the other hand, in SHRs, the GYY4137 showed no effect on ICI even at the same doses and SHR bladder strips showed NaHS-induced hypo-relaxation (BL-D) or lower potency in the NaHS-induced relaxation (BL-T) compared with the Wistar rat strips.  These data suggest that intravesical H2S-induced suppression of the micturition reflex might be impaired in SHRs at least by attenuating H2S-induced relaxation of the bladder smooth muscle.
Compared with normotensive Wistar rat bladder tissues, higher H2S content and an increase in MPST protein expression were detected in the SHR bladder tissues.  These data indicate that the endogenous H2S system might be upregulated to compensate the attenuated relaxation response to H2S in the SHR bladder.  Enhancement of the bladder afferent activity, a cause of OAB, is reported in SHRs.  Because it is reported that H2S promotes the release of sensory neuropeptides from the bladder afferents to mediate inhibitory neurotransmission to the pig bladder neck, the H2S system might be upregulated to argument the inhibitor signal, thereby counteracting the enhanced bladder activity.

Our present data suggest that H2S-induced bladder relaxation in SHRs is impaired, which might be a cause of hypertension-mediated development of OAB-like bladder dysfunction.  To compensate the less responsiveness, endogenous H2S levels might be increased in the SHR bladder.  Therefore, the endogenous H2S system might be a new drug candidate for the treatment of hypertension-mediated OAB.

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