Hydrogen sulfide (H2S) is a famous toxic gas, but since a possible role of H2S as an endogenous neuromodulator in the brain was reported in 1996 [1], this molecule has been recognized as an endogenous gasotransmitter.  H2S has a lot of physiological functions including neuromodulation, smooth muscle relaxation, cytoprotection and regulation of insuline secretion.  We previously reported that H2S is a possible relaxation factor in the rat bladder [2], while roles of H2S in the brain in regulation of the micturition reflex remains unclear.  

In the brain, mainly two mechanisms for H2S biosynthesis are reported; (1) a cystathionine β-synthase (CBS)-mediated mechanism and (2) a cysteine aminotraosferase (CAT)/3-mercaptopyruvate sulfurtransferase (MPST)-mediated mechanism [3].  CBS synthesizes H2S from L-cysteine, while 3-MPST synthesizes H2S from 3-mercaptopyruvate, which is synthesized from L-cysteine by CAT.  In this study, to elucidate roles of brain H2S in regulation of the micturition reflex, we investigated effects of centrally administered GYY4137 (a H2S donor) and AOAA (a non-selective inhibitor of CBS and CAT) on the rat micturition reflex.

Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (350-400 g) were used.  A catheter was inserted into the bladder from the bladder dome to perform cystometry.  Two hours after the surgery, intravesical instillation of saline at 12 ml/h was started to evaluate intercontraction intervals (ICI) and maximal voiding pressure (MVP).  One hour after the start, GYY4137 (3 or 10 nmol/rat), AOAA (30 or 100 µg/rat) or vehicle was intracerebroventricularly administered.  Evaluations of ICI and MVP were continued 90 min after the administration.  In some rats, through a catheter inserted into the femoral vein, GYY4137 (10 nmol/rat) or AOAA (30 µg/rat) was intravenously administered instead of intracerebroventricular administration.  We also performed single cystometry (saline instillation rate at 12 ml/h).  After 4-5 times of single cystometry, AOAA (30 µg/rat) was intracerebroventricularly administered, then single cystometry was continued for 30 min.

Effects of intracerebroventricular pretreatment with GYY4137 (10 nmol/rat) on intracerebroventricularly administered AOAA (30 µg/rat)-induced responses were investigated.  In addition, we also investigated effects of intracerebroventricularly pretreated SR95531 (SR, a GABAA receptor antagonist, 0.1 nmol in 5 µl saline/rat) and SCH50911 (SCH, a GABAB receptor antagonist, 0.1 nmol in 5 µl saline/rat) on the intracerebroventricularly administered GYY4137 (10 nmol/rat)-induced responses.  Each pretreatment was performed 30 min before AOAA or GYY4137 administration.

Intracerebroventricularly administered GYY4137 dose-dependently prolonged ICI and intracerebroventricularly administered AOAA significantly shortened ICI (Fig. 1A and 1B), withoug changing MVP (data not shown).  On the other hand, intravenously administered GYY4137 or AOAA showed no significant effect on ICI or MVP (data not shown).  Intracerebroventricularly administered AOAA significantly reduced single-voided volume (Vv) and bladder capacity (BC) without affecting post-voiding residual volume (Rv) or voiding efficiency (VE) [relative values of these parameters to baseline values before AOAA administration were as follows (means±SEM); Vv, 70.5±8.5%; Rv, 81.2±23.3%; BC, 75.0±9.8%; VE, 99.7±5.9% (n=7)].  Intracerebroventricularly administered AOAA-induced ICI shortening was cancelled by intracerebroventricular pretreatment with GYY4137 (Fig. 1C).  ICI prolongation induced by intracerebroventricularly administered GYY4137 was significantly attenuated by intracerebroventricularly pretreated SR and SCH (Fig. 2A and 2B).

GYY4137-induced ICI prolongation was induced by intracerebroventricular, but not intravenous, administration, indicating that H2S derived from GYY4137 centrally suppressed the micturition reflex.  AOAA-induced ICI shortening was induced by intracerebroventricular, but not intravenous, administration, and intracerebroventricularly administered AOAA induced reduction in Vv and BC without altering Rv or VE.  These data suggest that AOAA-mediated inhibition of H2S biosynthesis in the brain induced frequent urination.  In fact, supplementation of H2S by intracerebroventricular pretreatment with GYY4137 cancelled the intracerebroventricularly administered AOAA-induced ICI shortening, indicating that brain endogenous H2S play a suppressive role in regulation of the micturition reflex.  It is reported that an H2S donor recovered down-regulated GABAB receptor subunits in the hippocaupus of recurrent febrile seizures rats, and that H2S enhanced GABAA receptor-mediated baroreceptor function in spontanously hypertensive rats.  These findings suggest that H2S can modulate GABAergic neurotransmission.  In this study, intracerebroventricular pretreatment with SR or SCH respectively attenuated the intracerebroventricularly administered GYY4137-induced ICI prolongation.  Therefore, H2S derived from GYY4137 in the brain can suppress the micturition reflex through brain GABAA and GABAB receptors.

Endogenous H2S in the brain centrally suppresses the micturition reflex through brain GABAA and GABAB receptors in rats.  Thus, brain H2S could be a novel therapeutic target for patients who cannot obtain sufficient therapeutic effects from conventional medications on lower urinary tract dysfunctions.

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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.
Zhang X, Bian JS. Hydrogen sulfide: a neuromodulator and neuroprotectant in the central nervous system. ACS Chem Neurosci. 2014;5:876-883.