Nicotinic acetylcholine receptors (nAChRs) play an important role in regulation of the lower urinary tract (LUT) through synaptic transmission in the autonomic ganglia and neuromuscular junction in urethral striated muscle. Functions of the LUT are also regulated by the brain in coordinating with the peripheral nervous system and the spinal cord. Previously, we reported that activation of brain α7 nAChRs, an abundant subtype in the brain, induced prolongation of intercontraction intervals (ICI) through the brain GABAergic nervous system in rats [1]. 
    We previously found that the brain GABAergic nervous system is involved in the inhibition of the rat micturition reflex induced by a centrally administered donor of hydrogen sulfide (H2S) [2], an endogenous gasotransmitter. The production of endogenous H2S can be modulated by nAChR activation [3]; however, the relationship between α7 nAChRs and H2S in the brain during inhibition of the micturition reflex remains unclear. Therefore, we investigated whether H2S in the brain is involved in the inhibition of the micturition reflex induced by activation of α7 nAChRs in rats.

Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (350-420 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 ICI and maximal voiding pressure (MVP). One hour after the start, GYY4137 (an H2S donor, 1 or 3 nmol/rat) AOAA (a non-selective inhibitor of H2S synthesis, 3 or 10 µg) or vehicle was intracerebroventricularly (icv) pre-treated. Subsequently, PHA568487 (PHA, an α7 nAChR agonist, 0.3 or 1 nmol/rat) was icv administered 30 min (AOAA) or 60 min (GYY4137) after each pre-treatment. When both GYY4137 and AOAA were pre-treated, GYY4137 (1 nmol/rat) was icv pre-treated 30 min before AOAA administration (10 µg/rat, icv), and PHA (1 nmol/rat) was icv administered 30 min after AOAA pre-treatment. Evaluations of ICI and MVP were continued 1 h after the PHA administration.

Consistent with our previous findings [1], PHA at a lower dose (0.3 nmol/rat, icv) did not influence on ICI or MVP (Fig. A). In contrast, under central pre-treatment with GYY4137 (3 nmol/rat, icv), PHA markedly prolonged ICI even at a lower dose (0.3 nmol/rat, icv) compared with that in the vehicle (1% DMF)-pre-treated group (Fig. A). There was no significant difference in the MVP among the three group (data not shown). Preliminary, we confirmed that GYY4137 alone had no significant effect on the ICI or MVP at each dose (data not shown), which is consistent with our previous findings [2].
    PHA at a higher dose (1 nmol/rat, icv) prolonged ICI (Fig. B) without affecting MVP (data not shown) [1], consistent with our previous findings [1]. Central pre-treatment with AOAA (10 µg/rat, icv) markedly suppressed the PHA-induced response (Fig. B). There was no significant difference in the MVP among the three group (data not shown). Preliminary, we confirmed that AOAA alone had no significant effect on the ICI or MVP at each dose (data not shown), which is consistent with our previous findings [2].
    Central pre-treatment with AOAA (10 μg/rat, icv) significantly suppressed the PHA (1 nmol/rat, icv)-induced ICI prolongation, whereas the AOAA-induced suppression was significantly annulled under central presence of GYY4137 (1 nmol/rat, icv) (Fig. C).

At first, we used GYY4137 and PHA at an ineffective dose to induce ICI prolongation (GYY4137 at 3 nmol/rat; PHA at 0.3 nmol/rat). However, centrally pre-treated GYY4137 and icv administered PHA568487 induced ICI prolongation. These results suggest that centrally pre-treated GYY4137 potentiated the centrally administered PHA-induced inhibition of the micturition reflex. Therefore, H2S in the brain might be involved in the inhibition of the micturition reflex induced by activation of α7 nAChRs in rats.
    Next, to clarify the roles of endogenous H2S in the brain during PHA-induced ICI prolongation, we used AOAA, a non-selective inhibitor of H2S synthesis. In a previous study, centrally administered AOAA (30 and 100 μg/rat, icv) shortened the ICI in rats [2]. Thus, we used AOAA at ineffective dose to shorten the ICI (3 and 10 μg/rat). We found that icv pre-treated AOAA suppressed PHA-induced ICI prolongation at an effective dose (1 nmol/rat). These results suggest that centrally pre-treated AOAA suppressed the centrally administered PHA-induced inhibition of the micturition reflex.
    Owing to the lesser selectivity of AOAA to inhibit H2S synthesis, we confirmed whether centrally administered AOAA-induced suppression of the PHA-induced ICI prolongation was mediated through AOAA-induced inhibition of endogenous H2S production. To validate this, we investigated the effects of central supplementation of H2S induced by GYY4137 on the AOAA-induced suppression. In this experiment, we used AOAA at an effective dose (10 μg/rat) to inhibit the PHA-induced response and GYY4137 at an ineffective dose (1 nmol/rat) to potentiate the PHA-induced response. The AOAA-induced suppression of the PHA-induced ICI prolongation was negated by centrally pre-treated GYY4137, suggesting that the AOAA-induced suppression may be negated by the GYY4137-induced central supplementation of H2S. Therefore, endogenous H2S in the brain can be involved in the inhibition of the micturition reflex induced by activation of α7 nAChRs in rats.

H2S is involved in α7 nAChR-mediated inhibition of the rat micturition reflex in the brain. Therefore, brain H2S and α7 nAChRs could be novel therapeutic targets for patients with LUT dysfunctions attributed to neurogenic bladder overactivity.

Shimizu Y, Shimizu T, Zou S, et al. Stimulation of brain ?7-nicotinic acetylcholine receptors suppresses the rat micturition through brain GABAergic receptors. Biochem Biophys Res Commun. 2021; 548: 84-90.
Yamamoto M, Shimizu T, Zou S, et al. Brain hydrogen sulfide suppresses the micturition reflex via brain GABA receptors in rats. Nitric Oxide. 2020; 104-105: 44-50.
Wilinski J, Wilinski B, Somogyi E, et al. Nicotine affects hydrogen sulfide concentrations in mouse kidney and heart but not in brain and liver tissues. Folia Med Cracov. 2017; 57: 55-64.

Continence 7S1 (2023) 100963
DOI: 10.1016/j.cont.2023.100963