Hypothesis / aims of study
The central cholinergic system is reported to suppress the micturition reflex through brain muscarinic acetylcholine (ACh) receptors. ACh also stimulates nicotinic ACh receptors (nAChRs), which are reported to suppress the micturition reflex in the brain. It was previously reported that activation of brain α7 nAChRs, an abundant subtype in the brain, suppressed the rat micturition reflex via brain hydrogen sulfide (H2S) [1,2], an endogenous gasotransmitter. Because H2S is reported to show a synergic action with another endogenous gasotransmitter nitric oxide (NO) [3], we investigated roles of brain NO in suppression of the micturition reflex induced by activation of brain α7 nAChRs in rats.
Study design, materials and methods
Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (300-500 g) were used.
(1) A catheter was inserted into the bladder from the bladder dome to perform continuous cystometry. Two hours after the surgery, intravesical instillation of saline at 12 ml/h was started to evaluate intercontraction interval (ICI) and maximal voiding pressure (MVP). One hour after the start, SNAP (an NO donor, 10 or 30 nmol/rat), L-NAME [a non-selective inhibitor of NO synthase (NOS), 30 or 100 nmol/rat] or vehicle was intracerebroventricularly (icv) administered. Evaluations of ICI and MVP were continued 2 h after the administration.
(2) Three hours after the surgery described in (1), single cystometry was performed. After 5 times of single cystometry, SNAP (30 nmol/rat) was icv administered, then single cystometry was performed (intravesical instillation of saline at 12 ml/h) during 30-90 min after the administration.
(3) Two hours after the surgery described in (1), intravesical instillation of saline at 12 ml/h was started to evaluate ICI and MVP. One hour after the start, SNAP (3 or 10 nmol/rat), L-NAME (10 or 30 nmol/rat) or vehicle was icv pre-treated. Subsequently, PHA568487 (PHA, α7 nAChR agonist, 0.3 or 1 nmol/rat) was icv administered 60 min (SNAP) or 30 min (L-NAME) after each pre-treatment. Evaluations of ICI and MVP were continued 1 h after the PHA administration.
Results
(1) Centrally administered SNAP at 30 nmol/rat significantly shortened ICI (Fig. 1A) without changing MVP (data not shown). On the other hand, centrally administered L-NAME at 100 nmol/rat significantly prolonged ICI (Fig. 1B) without changing MVP (data not shown).
(2) Centrally administered SNAP (30 nmol/rat) significantly reduced single-voided volume (Vv) and bladder capacity (BC) without affecting post-voided residual volume (Rv) or voiding efficiency (VE) (Table 1).
(3) Consistent with previous findings [1], PHA at a higher dose (1 nmol/rat, icv) prolonged ICI (Fig. 2A) without affecting MVP (data not shown). Central pre-treatment with SNAP at 10 nmol/rat, which showed no effect on ICI (Fig. 1A), significantly suppressed the PHA-induced ICI prolongation (Fig. 2A). There was no significant difference in the MVP among the three group (data not shown).
PHA at a lower dose (0.3 nmol/rat, icv) did not influence on ICI (Fig. 2B) or MVP (data not shown), consistent with previous findings [1]. In contrast, under central pre-treatment with L-NAME at 30 nmol/rat, which showed no effect on ICI (Fig. 1B), PHA significantly prolonged ICI even at a lower dose (0.3 nmol/rat, icv) (Fig. 2B). There was no significant difference in the MVP among the three group (data not shown).
Interpretation of results
In this study, centrally administered SNAP shortened ICI and reduced Vv and BC without altering MVP, Rv or VE, suggesting that SNAP-derived NO in the brain induced frequent urination. On the other hand, centrally administered L-NAME prolonged ICI without altering MVP, indicating that L-NAME-mediated inhibition of NO biosynthesis in the brain centrally suppressed the micturition reflex. Therefore, brain endogenous NO can play a facilitative role in regulation of the micturition reflex.
Next, we attempted to clarify the roles of brain NO during PHA-induced ICI prolongation using SNAP. In our data, centrally administered SNAP (30 nmol/rat, icv) shortened ICI in rats (Fig. 1A). Thus, we pre-treated SNAP at ineffective doses to shorten the ICI (3 and 10 nmol/rat). We found that icv pre-treated SNAP suppressed the PHA (1 nmol/rat, icv)-induced ICI prolongation. These results suggest that centrally pre-treated SNAP-derived NO inhibited the centrally administered PHA-induced suppression of the micturition reflex.
Furthermore, we used L-NAME and PHA at an ineffective dose to induce ICI prolongation (L-NAME at 30 nmol/rat; PHA at 0.3 nmol/rat). Under central pre-treatment with L-NAME, PHA induced ICI prolongation even at the ineffective dose. These results suggest that centrally pre-treated L-NAME-mediated inhibition of NO biosynthesis in the brain potentiated the centrally administered PHA-induced suppression of the micturition reflex. Therefore, endogenous NO in the brain can play an inhibitory role in suppression of the micturition reflex induced by activation of brain α7 nAChRs.
It is reported a functional relationship between nAChRs and NO signaling, therefore, brain NO might play a brake-like role to prevent excessive suppression of the micturition reflex via brain α7 nAChRs. Although NO is reported to show synergic vasodilation with H2S [3], H2S is involved in suppression of the micturition reflex induced by activation of brain α7 nAChRs [2]. Thus, unlike vessel, brain NO and H2S might antagonistically regulate the brain α7 nAChR-mediated suppression of the micturition reflex.