Hydrogen sulfide (H2S), an endogenous gasotransmitter, has a wide range of physiological functions including neuromodulation, vasorelaxation and cytoprotection.  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).  We previously reported that (1) at least the CAT/MPST pathway-mediated endogenous H2S production was working in the rat bladder tissues, (2) NaHS, an H2S donor, induced relaxation of pre-contracted the rat bladder tissue strips and (3) intravesically instilled GYY4137, an H2S donor, suppressed the rat micturition reflex [1].  These findings indicate a possibility that H2S can function as an endogenous relaxation factor in the rat bladder.
Recently, bladder ischemia induced by chronic hypertension has been recognized as an etiologic factor of lower urinary tract dysfunctions including overactive bladder and detrusor overactivity (DO).  The spontaneously hypertensive rat (SHR) develops DO and shows frequent urination compared with a normotensive control.  We recently reported that H2S donors-induced bladder relaxation and suppression of the micturition reflex were attenuated in SHRs at 18 weeks of age compared with age-matched normotensive Wistar rats [2].  On the other hand, SHRs are reported to show DO after a certain age, at lease at 17 and 21 weeks of age but not at 12 weeks of age [3] although hypertension is already detected at each age, indicating that hypertension-mediated bladder dysfunctions may be detected after hypertension is developed.  Therefore, we hypothesized that responses to exogenous H2S and levels of the endogenous H2S system in the SHR bladder might be different with age.  In this study, we compared effects of GYY4137 and NaHS on the micturition reflex and on the bladder contractility, and the endogenous H2S system in the bladder of SHRs at between 12 and 18 weeks of age (12W and 18W).

Male SHRs at 12W and 18W 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) Bladder dome (BL-D) and trigone (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 intercontraction intervals (ICI) (sec, means ± SEM) just before intravesical instillation of vehicle or GYY4137 were 916±75 at 12W (n=16) and 1324±135 at 18W (n=14).  The values of SHRs at 12W were significantly shorter than those at 18W (P<0.05).  GYY4137 significantly prolonged ICI compared to the vehicle-treated group at 12W, but not at 18W (Fig. 1).
(2) NaHS-induced relaxation on pre-contracted BL-D and BL-T strips was significantly attenuated in SHRs at 18W compared with SHRs at 12W (Table 1).  The H2S content in the BL-D of SHRs at 18W was significantly higher than that at 12W (Fig. 2A).  CBS, MPST and CAT, but not CSE, were detected in the bladder of SHRs at both ages (Fig. 2B).  The expression levels of CBS, MPST and CAT in the SHR BL-D at 18W were significantly higher than those at 12W (Fig. 2B).

Basal values of ICI in SHRs at 18W was prolonged compared with those at 12W in line with a previous report by another group [3].  The group also reported higher bladder capacity and single voided volume in SHRs at 18W than those at 12W [3].  These lines of evidence suggest that SHRs might begin to show chronic hypertension-mediated morphological changes such as hypertrophy in the bladder after a certain age.   Intravesically instilled GYY4137 prolonged ICI in SHRs at 12W and NaHS relaxed pre-contracted bladder strips of SHRs at 12W, while these H2S donors-induced changes were not detected in SHRs at 18W.  These results suggest that the GYY4137-induced inhibition of the micturition reflex in SHRs at 12W might be mediated at least by relaxation of the bladder smooth muscle, and sensitivity to H2S-induced bladder relaxation in SHRs might have a close relationship with aging.  Compared with bladder tissues from SHRs at 12W, higher H2S content and increased protein expression of CBS, MPST and CAT were detected in bladder tissues from SHRs at 18W.  These data indicate that the endogenous H2S system might be upregulated to compensate the attenuated relaxation response to H2S in the SHR bladder after a certain age.

H2S-induced bladder relaxation in SHRs can be impaired in an age-dependent manner, and to compensate the less responsiveness, endogenous H2S levels are increased in the SHR bladder after a certain age.  Therefore, early intervention to the endogenous H2S system in SHRs may be prevent the development of hypertension-mediated bladder dysfunctions.

Zou S, Shimizu T, Shimizu S, et al. Possible role of hydrogen sulfide as an endogenous relaxation factor in the rat bladder and prostate. Neurourol Urodyn. 2018;37:2519-2526.
Zou S, Shimizu T, Yamamoto M, et al. Hydrogen sulfide-induced relaxation of the bladder is attenuated in spontaneously hypertensive rats. Int Urol Nephrol. 2019;51:1507-1515.
Jin JH, Lee HJ, Shin HY et al. Development and changes with age of detrusor overactivity in spontaneous hypertensive rats as observed by simultaneous registrations of intravesical and intraabdominal pressures. Int Neurourol J. 2011;15:192-198.